Insulin derivatives

ABSTRACT

The present invention relates to insulin derivatives which are naturally occurring insulins or analogues thereof which have a side chain attached either to the α-amino group of the N-terminal amino acid residue of the B chain or to the ε-amino group of a Lys residue present in the B chain of the parent insulin, the side chain being of the general formula:
 
—W—X—Y—Z
 
wherein W, X, Y and Z are as defined in the disclosure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/343,005, filed Jan. 30, 2006, which is a continuation ofInternational Application No. PCT/DK2004/000511, filed Jul. 22, 2004,which claims priority from Danish Patent Application No. PA 2003 01129filed Aug. 5, 2003 and to U.S. patent application Ser. No. 60/495,451filed Aug. 14, 2003.

FIELD OF THE INVENTION

The present invention relates to novel human insulin derivatives whichare soluble at physiological pH values and have a prolonged profile ofaction. The invention also relates to methods of providing suchderivatives, to pharmaceutical compositions containing them, to a methodof treating diabetes and hyperglycaemia using the insulin derivatives ofthe invention and to the use of such insulin derivatives in thetreatment of diabetes and hyperglycaemia.

BACKGROUND OF THE INVENTION

Currently, the treatment of diabetes, both type 1 diabetes and type 2diabetes, relies to an increasing extent on the so-called intensiveinsulin treatment. According to this regimen, the patients are treatedwith multiple daily insulin injections comprising one or two dailyinjections of a long acting insulin to cover the basal insulinrequirement supplemented by bolus injections of a rapid acting insulinto cover the insulin requirement related to meals.

Long acting insulin compositions are well known in the art. Thus, onemain type of long acting insulin compositions comprises injectableaqueous suspensions of insulin crystals or amorphous insulin. In thesecompositions, the insulin compounds utilized typically are protamineinsulin, zinc insulin or protamine zinc insulin.

Certain drawbacks are associated with the use of insulin suspensions.Thus, in order to secure an accurate dosing, the insulin particles mustbe suspended homogeneously by gentle shaking before a defined volume ofthe suspension is withdrawn from a vial or expelled from a cartridge.Also, for the storage of insulin suspensions, the temperature must bekept within more narrow limits than for insulin solutions in order toavoid lump formation or coagulation.

While it was earlier believed that protamines were non-immunogenic, ithas now turned out that protamines can be immunogenic in man and thattheir use for medical purposes may lead to formation of antibodies.Also, evidence has been found that the protamine-insulin complex isitself immunogenic. Therefore, with some patients the use of long actinginsulin compositions containing protamines must be avoided.

Another type of long acting insulin compositions are solutions having apH value below physiological pH from which the insulin will precipitatebecause of the rise in the pH value when the solution is injected. Adrawback with these solutions is that the particle size distribution ofthe precipitate formed in the tissue on injection, and thus the releaseprofile of the medication, depends on the blood flow at the injectionsite and other parameters in a somewhat unpredictable manner. A furtherdrawback is that the solid particles of the insulin may act as a localirritant causing inflammation of the tissue at the site of injection.

WO 91/12817 (Novo Nordisk A/S) discloses soluble insulin compositionscomprising insulin complexes of cobalt(III). The action profile of thesecomplexes is only moderately prolonged and the bioavailability isreduced relative to human insulin.

Human insulin has three primary amino groups: the N-terminal group ofthe A-chain and of the B-chain and the ε-amino group of Lys^(B29).Several insulin derivatives which are substituted in one or more ofthese groups are known in the prior art. Thus, U.S. Pat. No. 3,528,960(Eli Lilly) relates to N-carboxyaroyl insulins in which one, two orthree primary amino groups of the insulin molecule has a carboxyaroylgroup.

According to GB Patent No. 1.492.997 (Nat. Res. Dev. Corp.), it has beenfound that insulin with a carbamyl substitution at N^(εB29) has animproved profile of hypoglycaemic effect.

JP laid-open patent application No. 1-254699 (Kodama Co., Ltd.)discloses insulin wherein a fatty acid is bound to the amino group ofPhe^(B1) or to the ε-amino group of Lys^(B29) or to both of these. Thestated purpose of the derivatisation is to obtain a pharmacologicallyacceptable, stable insulin preparation.

Insulins, which in the B30 position have an amino acid having at leastfive carbon atoms which cannot necessarily be coded for by a triplet ofnucleotides, are described in JP laid-open patent application No.57-067548 (Shionogi). The insulin analogues are claimed to be useful inthe treatment of diabetes mellitus, particularly in patients who areinsulin resistant due to generation of bovine or porcine insulinantibodies.

WO 95/07931 (Novo Nordisk A/S) discloses human insulin derivativeswherein the ε-amino group of Lys^(B29) has a lipophilic substituent.These insulin derivatives have a prolonged profile of action and aresoluble at physiological pH values.

EP 894095 discloses insulin derivatives wherein the N-terminal group ofthe B-chain and/or the ε-amino group of Lys in position B28, B29 or B30has a substituent of the formula —CO—W—COOH where W can be a long chainhydrocarbon group. These insulin derivatives have a prolonged profile ofaction and are soluble at physiological pH values.

However, there is still a need for insulins having a more prolongedprofile of action than the insulin derivatives known up till now andwhich at the same time are soluble at physiological pH values and have apotency which is comparable to that of human insulin.

SUMMARY OF THE INVENTION

The present invention is based on the recognition that the overallhydrophobicity of an insulin derivative molecule plays an important rolefor the in vivo potency of the derivative.

In one aspect the present invention relates to an insulin derivativewhich is a naturally occurring insulin or an analogue thereof which hasa side chain attached either to the α-amino group of the N-terminalamino acid residue of the B chain or to the ε-amino group of a Lysresidue present in the B chain of the parent insulin, the side chainbeing of the general formula:—W—X—Y—Zwherein W is:

-   -   an α-amino acid residue having a carboxylic acid group in the        side chain which residue forms, with one of its carboxylic acid        groups, an amide group together with the α-amino group of the        N-terminal amino acid residue of the B chain or together with        the ε-amino group of a Lys residue present in the B chain of the        parent insulin;    -   a chain composed of two, three or four α-amino acid residues        linked together via amide bonds, which chain—via an amide        bond—is linked to the α-amino group of the N-terminal amino acid        residue of the B chain or to the ε-amino group of a Lys residue        present in the B chain of the parent insulin, the amino acid        residues of W being selected from the group of amino acid        residues having a neutral side chain and amino acid residues        having a carboxylic acid group in the side chain so that W has        at least one amino acid residue which has a carboxylic acid        group in the side chain; or    -   a covalent bond from X to the α-amino group of the N-terminal        amino acid residue of the B chain or to the ε-amino group of a        Lys residue present in the B chain of the parent insulin;        X is:    -   —CO—;    -   —CH(COOH)CO—;    -   —N(CH₂COOH)CH₂ CO—;    -   —N(CH₂COOH)CH₂CON(CH₂COOH)CH₂ CO—;    -   —N(CH₂CH₂COOH)CH₂CH₂ CO—;    -   —N(CH₂CH₂COOH)CH₂CH₂CON(CH₂CH₂COOH)CH₂CH₂ CO—;    -   —NHCH(COOH)(CH₂)₄NHCO—;    -   —N(CH₂CH₂COOH)CH₂ CO—; or    -   —N(CH₂COOH)CH₂CH₂ CO—.        that

-   a) when W is an amino acid residue or a chain of amino acid    residues, via a bond from the underscored carbonyl carbon forms an    amide bond with an amino group in W, or

-   b) when W is a covalent bond, via a bond from the underscored    carbonyl carbon forms an amide bond with the N-terminal α-amino    group in the B chain or with the ε-amino group of a Lys residue    present in the B chain of the parent insulin;    Y is:    -   —(CH₂)_(m)— where m is an integer in the range of 6 to 32;    -   a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups        and a number of —CH₂— groups sufficient to give a total number        of carbon atoms in the chain in the range of 10 to 32;    -   a divalent hydrocarbon chain of the formula        —(CH₂)_(v)C₆H₄(CH₂)_(w)— wherein v and w are integers or one of        them is zero so that the sum of v and w is in the range of 6 to        30; and        Z is:    -   —COOH;    -   —CO-Asp;    -   —CO-Glu;    -   —CO-Gly;    -   —CO-Sar;    -   —CH(COOH)₂;    -   —N(CH₂COOH)₂;    -   —SO₃H; or    -   —PO₃H;        and any Zn²⁺ complexes thereof, provided that when W is a        covalent bond and X is —CO—, then Z is different from —COOH.

In one embodiment of the invention, the side chain —W—X—Y—Z is attachedto the α-amino group of the N-terminal amino acid residue of the B chainof the parent insulin.

In another embodiment of the invention, side chain —W—X—Y—Z is attachedto the ε-amino group of a Lys residue present in the B chain of theparent insulin. In one more specific aspect of this embodiment, the sidechain —W—X—Y—Z is attached to the ε-amino group of a Lys residue presentin position 28 of the B chain. In a further more specific aspect of thisembodiment, the side chain —W—X—Y—Z is attached to the ε-amino group ofa Lys residue present in position 29 of the B chain. In a further morespecific aspect of this embodiment, the side chain —W—X—Y—Z is attachedto the ε-amino group of a Lys residue present in position 30 of the Bchain.

The substructure W of the side chain —W—X—Y—Z can be a covalent bond.Alternatively, W can be a residue of an α-amino acid having a carboxylicacid group in the side chain and comprising a total of from 4 to 10carbon atoms. Specifically, W can be the residue of an α-amino acid,that can be coded for by the genetic code. Thus, W can, for example, beselected from the group consisting of α-Asp, β-Asp, α-Glu, and γ-Glu.Further options for W are for example α-hGlu and δ-hGlu.

In a further embodiment, W is a chain composed of two α-amino acidresidues of which one has from 4 to 10 carbon atoms and a carboxylicacid group in the side chain while the other has from 2 to 11 carbonatoms but no free carboxylic acid group. The α-amino acid residue withno free carboxylic acid group can be a neutral, codable α-amino acidresidue. Examples of W according to this embodiment are: α-Asp-Gly;Gly-α-Asp; β-Asp-Gly; Gly-β-Asp; α-Glu-Gly; Gly-α-Glu; γ-Glu-Gly;Gly-γ-Glu; α-hGlu-Gly; Gly-α-hGlu; δ-hGlu-Gly; and Gly-δ-hGlu.

In a further embodiment, W is a chain composed of two α-amino acidresidues, independently having from 4 to 10 carbon atoms, and bothhaving a carboxylic acid group in the side chain. One of these α-aminoacid residues or both of them can be codable α-amino acid residues.Examples of W according to this embodiment are: α-Asp-α-Asp;α-Asp-α-Glu; α-Asp-α-hGlu; α-Asp-β-Asp; α-Asp-γ-Glu; α-Asp-δ-hGlu;β-Asp-α-Asp; β-Asp-α-Glu; β-Asp-α-hGlu; β-Asp-β-Asp; β-Asp-γ-Glu;β-Asp-δ-hGlu; α-Glu-α-Asp; α-Glu-α-Glu; α-Glu-α-hGlu; α-Glu-β-Asp;α-Glu-γ-Glu; α-Glu-δ-hGlu; γ-Glu-α-Asp; γ-Glu-α-Glu; γ-Glu-α-hGlu;γ-Glu-β-Asp; γ-Glu-γ-Glu; γ-Glu-δ-hGlu; α-hGlu-α-Asp; α-hGlu-α-Glu;α-hGlu-α-hGlu; α-hGlu-β-Asp; α-hGlu-γ-Glu; α-hGlu-δ-hGlu; δ-hGlu-α-Asp;δ-hGlu-α-Glu; δ-hGlu-α-hGlu; δ-hGlu-β-Asp; δ-hGlu-γ-Glu; andδ-hGlu-δ-hGlu.

In a further embodiment, W is a chain composed of three α-amino acidresidues, independently having from 4 to 10 carbon atoms, the amino acidresidues of the chain being selected from the group of residues having aneutral side chain and residues having a carboxylic acid group in theside chain so that the chain has at least one residue which has acarboxylic acid group in the side chain. In one embodiment, the aminoacid residues are codable residues.

In a further embodiment, W is a chain composed of four α-amino acidresidues, independently having from 4 to 10 carbon atoms, the amino acidresidues of the chain being selected from the group having a neutralside chain and residues having a carboxylic acid group in the side chainso that the chain has at least one residue which has a carboxylic acidgroup in the side chain. In one embodiment, the amino acid residues arecodable residues.

In one embodiment W can be connected to the ε-amino group of the Lysresidue in the B-chain via an urea derivative.

The substructure X of the side chain —W—X—Y—Z can be a group of theformula —CO— that, via a bond from the underscored carbonyl carbon,forms an amide bond with an amino group in W or, when W is a covalentbond, with the N-terminal α-amino group in the B chain or with theε-amino group of a Lys residue present in the B chain of the parentinsulin.

In a further embodiment, the substructure X of the side chain can be agroup of the formula —CH(COOH)CO— that, via a bond from the underscoredcarbonyl carbon, forms an amide bond with an amino group in W or, when Wis a covalent bond, with the N-terminal α-amino group in the B chain orwith the ε-amino group of a Lys residue present in the B chain of theparent insulin.

In a further embodiment, the substructure X of the side chain can be agroup of the formula —N(CH₂COOH)CH₂ CO— that, via a bond from theunderscored carbonyl carbon, forms an amide bond with an amino group inW or, when W is a covalent bond, with the N-terminal α-amino group inthe B chain or with the ε-amino group of a Lys residue present in the Bchain of the parent insulin.

In a further embodiment, the substructure X of the side chain can be agroup of the formula —N(CH₂CH₂COOH)CH₂ CO— that, via a bond from theunderscored carbonyl carbon, forms an amide bond with an amino group inW or, when W is a covalent bond, with the N-terminal α-amino group inthe B chain or with the ε-amino group of a Lys residue present in the Bchain of the parent insulin.

In a further embodiment, the substructure X of the side chain can be agroup of the formula —N(CH₂COOH)CH₂ CH₂ CO— that, via a bond from theunderscored carbonyl carbon, forms an amide bond with an amino group inW or, when W is a covalent bond, with the N-terminal α-amino group inthe B chain or with the ε-amino group of a Lys residue present in the Bchain of the parent insulin.

In a further embodiment, the substructure X of the side chain can be agroup of the formula —N(CH₂COOH)CH₂CON(CH₂COOH)CH₂ CO— that, via a bondfrom the underscored carbonyl carbon, forms an amide bond with an aminogroup in W or, when W is a covalent bond, with the N-terminal α-aminogroup in the B chain or with the ε-amino group of a Lys residue presentin the B chain of the parent insulin.

In a further embodiment, the substructure X of the side chain can be agroup of the formula —N(CH₂CH₂COOH)CH₂CH₂ CO— that, via a bond from theunderscored carbonyl carbon, forms an amide bond with an amino group inW or, when W is a covalent bond, with the N-terminal α-amino group inthe B chain or with the ε-amino group of a Lys residue present in the Bchain of the parent insulin.

In a further embodiment, the substructure X of the side chain can be agroup of the formula —N(CH₂CH₂COOH)CH₂CH₂CON(CH₂CH₂COOH)CH₂CH₂ CO— that,via a bond from the underscored carbonyl carbon, forms an amide bondwith an amino group in W or, when W is a covalent bond, with theN-terminal α-amino group in the B chain or with the ε-amino group of aLys residue present in the B chain of the parent insulin.

The substructure Y of the side chain —W—X—Y—Z can be a group of theformula —(CH₂)_(m)— where m is an integer in the range of from 6 to 32,from 8 to 20, from 12 to 20, or from 12-16.

In another embodiment, Y is a divalent hydrocarbon chain comprising 1, 2or 3 —CH═CH— groups and a number of —CH₂— groups sufficient to give atotal number of carbon atoms in the chain in the range of from 6 to 32,from 10 to 32, from 12 to 20, or from 12-16.

In another embodiment, Y is a divalent hydrocarbon chain of the formula—(CH₂)_(v)C₆H₄(CH₂)_(w)— wherein v and w are integers or one of them iszero so that the sum of v and w is in the range of from 6 to 30, from 10to 20, or from 12-16.

In one embodiment, the substructure Z of the side chain —W—X—Y—Z is—COOH provided that when W is a covalent bond and X is —CO—, then Z isdifferent from —COOH.

In another embodiment, Z is —CO-Asp.

In another embodiment, Z is —CO-Glu.

In another embodiment, Z is —CO-Gly.

In another embodiment, Z is —CO-Sar.

In another embodiment, Z is —CH(COOH)₂.

In another embodiment, Z is —N(CH₂COOH)₂.

In another embodiment, Z is —SO₃H.

In another embodiment, Z is —PO₃H.

In a further embodiment W is selected from the group consisting ofα-Asp, β-Asp, α-Glu, and γ-Glu; X is —CO— or —CH(COOH)CO; Y is—(CH₂)_(m)— where m is an integer in the range of 12-18 and Z is —COOHor —CH(COOH)₂.

The insulin moiety—in the present text also referred to as the parentinsulin—of an insulin derivative according to the invention can be anaturally occurring insulin such as human insulin or porcine insulin.Alternatively, the parent insulin can be an insulin analogue.

In one group of parent insulin analogues, the amino acid residue atposition A21 is Asn.

In another group of parent insulin analogues, the amino acid residue atposition A21 is Gly. Specific examples from this group of analogues areGly^(A21) human insulin, Gly^(A21) des(B30)human insulin; andGly^(A21)Arg^(B31)Arg^(B32) human insulin.

In another group of parent insulin analogues, the amino acid residue atposition B1 has been deleted. A specific example from this group ofparent insulin analogues is des(B1) human insulin.

In another group of parent insulin analogues, the amino acid residue atposition B30 has been deleted. A specific example from this group ofparent insulin analogues is des(B30) human insulin.

In another group of parent insulin analogues, the amino acid residue atposition B28 is Asp. A specific example from this group of parentinsulin analogues is Asp^(B28) human insulin.

In another group of parent insulin analogues, the amino acid residue atposition B28 is Lys and the amino acid residue at position B29 is Pro. Aspecific example from this group of parent insulin analogues isLys^(B28)Pro^(B29) human insulin.

In another group of parent insulin analogues the amino acid residue inposition B30 is Lys and the amino acid residue in position B29 is anycodable amino acid except Cys, Met, Arg and Lys. An example is aninsulin analogue where the amino acid residue at position B29 is Thr andthe amino acid residue at position B30 is Lys. A specific example fromthis group of parent insulin analogues is Thr^(B29)Lys^(B30) humaninsulin.

In another group of parent insulin analogues, the amino acid residue atposition B3 is Lys and the amino acid residue at position B29 is Glu. Aspecific example from this group of parent insulin analogues isLys^(B3)Glu^(B29) human insulin.

Examples of insulin derivatives according to the invention are thefollowing compounds:

N^(εB29)—(N^(α)-(HOOC(CH₂)₁₄CO)-γ-Glu) des(B30) human insulin;

N^(εB29)—(N^(α)-(HOOC(CH₂)₁₅CO)-γ-Glu) des(B30) human insulin;

N^(εB29)—(N^(α)-(HOOC(CH₂)₁₆CO)-γ-Glu) des(B30) human insulin;

N^(εB29)—(N^(α)-(HOOC(CH₂)₁₇CO)-γ-Glu) des(B30) human insulin;

N^(εB29)—(N^(α)-(HOOC(CH₂)₁₈CO)-γ-Glu) des(B30) human insulin;

N^(εB29)—(N^(α)-(HOOC(CH₂)₁₆CO)-γ-Glu—N-(γ-Glu)) des(B30) human insulin;

N^(εB29)—(N^(α)-(Asp-OC(CH₂)₁₆CO)-γ-Glu) des(B30) human insulin;

N^(εB29)—(N^(α)-(Glu-OC(CH₂)₁₄CO)-γ-Glu) des(B30) human insulin;

N^(εB29)—(N^(α)-(Glu-OC(CH₂)₁₄CO—) des(B30) human insulin;

N^(εB29)—(N^(α)-(Asp-OC(CH₂)₁₆CO—) des(B30) human insulin;

N^(εB29)—(N^(α)-(HOOC(CH₂)₁₆CO)-α-Glu—N-(β-Asp)) des(B30) human insulin;

N^(εB29)—(N^(α)-(Gly-OC(CH₂)₁₃CO)-γ-Glu) des(B30) human insulin;

N^(εB29)—(N^(α)-(Sar-OC(CH₂)₁₃CO)-γ-Glu) des(B30) human insulin;

N^(εB29)—(N^(α)-(HOOC(CH₂)₁₃CO)-γ-Glu) des(B30) human insulin;

(N^(εB29)—(N^(α)-(HOOC(CH₂)₁₃CO)-β-Asp) des(B30) human insulin;

N^(εB29)—(N^(α)-(HOOC(CH₂)₁₃CO)-α-Glu) des(B30) human insulin;

N^(εB29)—(N^(α)-(HOOC(CH₂)₁₆CO)-γ-D-Glu) des(B30) human insulin;

N^(εB29)—(N^(α)-(HOOC(CH₂)₁₄CO)-β-D-Asp) des(B30) human insulin;

N^(εB29)—(N—HOOC(CH₂)₁₆CO-β-D-Asp) des(B30) human insulin;

N^(εB29)—(N—HOOC(CH₂)₁₄CO—IDA) des(B30) human insulin;

N^(εB29)—[N—(HOOC(CH₂)₁₆CO)—N-(carboxyethyl)-Gly] des(B30) humaninsulin;

N^(εB29)—[N—(HOOC(CH₂)₁₄CO)—N-(carboxyethyl)-Gly] des(B30) humaninsulin; and

N^(εB29)—[N—(HOOC(CH₂)₁₄CO)—N-(carboxymethyl)-β-Ala] des(B30) humaninsulin.

Insulin derivatives according to the invention may be provided in theform of essentially zinc free compounds or in the form of zinccomplexes. When zinc complexes of an insulin derivative according to theinvention are provided, two Zn²⁺ ions, three Zn²⁺ ions or four Zn²⁺ ionscan be bound to each insulin hexamer. Solutions of zinc complexes of theinsulin derivatives will contain mixtures of such species.

In a further aspect of the invention, a pharmaceutical compositioncomprising a therapeutically effective amount of an insulin derivativeaccording to the invention together with a pharmaceutically acceptablecarrier can be provided for the treatment of type 1 diabetes, type 2diabetes and other states that cause hyperglycaemia in patients in needof such a treatment. An insulin derivative according to the inventioncan be used for the manufacture of a pharmaceutical composition for usein the treatment of type 1 diabetes, type 2 diabetes and other statesthat cause hyperglycaemia.

In a further aspect of the invention, there is provided a pharmaceuticalcomposition for treating type 1 diabetes, type 2 diabetes and otherstates that cause hyperglycaemia in a patient in need of such atreatment, comprising a therapeutically effective amount of an insulinderivative according to the invention in mixture with an insulin or aninsulin analogue which has a rapid onset of action, together withpharmaceutically acceptable carriers and additives.

In one embodiment the invention provides a pharmaceutical compositionbeing a mixture of an insulin derivative according to the invention anda rapid acting insulin analogue selected group consisting of Asp^(B28)human insulin; Lys^(B28)Pro^(B29) human insulin and Lys^(B3)Glu^(B29)human insulin.

In one embodiment the invention provides a pharmaceutical compositioncomprising N^(εB29)—(N^(α)-(HOOC(CH₂)14CO)-γ-Glu) des(B30) human insulinand AspB28 human insulin together with pharmaceutically acceptablecarriers and additives.

The insulin derivative according to the invention and the rapid actinginsulin analogue can be mixed in a ratio from about 90/10%; about 70/30%or about 50/50%.

In a further aspect of the invention, there is provided a method oftreating type 1 diabetes, type 2 diabetes and other states that causehyperglycaemia in a patient in need of such a treatment, comprisingadministering to the patient a therapeutically effective amount of aninsulin derivative according to the invention together with apharmaceutically acceptable carrier and pharmaceutical acceptableadditives.

In a further aspect of the invention, there is provided a method oftreating type 1 diabetes, type 2 diabetes and other states that causehyperglycaemia in a patient in need of such a treatment, comprisingadministering to the patient a therapeutically effective amount of aninsulin derivative according to the invention in mixture with an insulinor an insulin analogue which has a rapid onset of action, together witha pharmaceutically acceptable carrier and pharmaceutical acceptableadditives.

In a further aspect, the present invention relates to insulinderivatives which have an overall hydrophobicity which is essentiallysimilar to that of human insulin.

In further aspect, the present invention relates to insulin derivativeswhich have a hydrophobic index, k′_(rel), which is in the range of fromabout 2 to about 200.

In a further aspect, the insulin derivatives of the present inventionhave a hydrophobic index, k′_(rel), which is in the range from about0.02 to about 10, from about 0.1 to about 5; from about 0.5 to about 5;or from about 0.5 to about 2.

According to one embodiment of the present invention the insulinderivatives will comprise a side chain —W—X—Y—Z as defined above whichhas at least one hydrophilic and at least one hydrophobic region.

According to another embodiment of the present invention, the insulinderivatives will comprise a side chain —W—X—Y—Z as defined above whichhas at least one free carboxylic acid group and according to a furtherembodiment, the side chain will have at least two free carboxylic acidgroups.

In another embodiment, the invention relates to a pharmaceuticalcomposition comprising an insulin derivative according to the inventionwhich is soluble at physiological pH values.

In another embodiment, the invention relates to a pharmaceuticalcomposition comprising an insulin derivative according to the inventionwhich is soluble at pH values in the interval from about 6.5 to about8.5.

In another embodiment, the invention relates to a pharmaceuticalcomposition with a prolonged profile of action which comprises aninsulin derivative according to the invention.

In another embodiment, the invention relates to a pharmaceuticalcomposition which is a solution containing from about 120 nmol/ml toabout 2400 nmol/ml, from about 400 nmol/ml to about 2400 nmol/ml, fromabout 400 nmol/ml to about 1200 nmol/ml, from about 600 nmol/ml to about2400 nmol/ml, or from about 600 nmol/ml to about 1200 nmol/ml of aninsulin derivative according to the invention or of a mixture of theinsulin derivative according to the invention with a rapid actinginsulin analogue.

Hydrophobicity Data on Insulin Derivatives According to the Invention.

The hydrophobicity (hydrophobic index) of the insulin derivatives of theinvention relative to human insulin, k′_(rel), was measured on aLiChrosorb RP18 (5 μm, 250×4 mm) HPLC column by isocratic elution at 40°C. using mixtures of A) 0.1 M sodium phosphate buffer, pH 7.3,containing 10% acetonitrile, and B) 50% acetonitrile in water aseluents. The elution was monitored by following the UV absorption of theeluate at 214 nm. Void time, t₀, was found by injecting 0.1 mM sodiumnitrate. Retention time for human insulin, t_(human), was adjusted to atleast 2t₀ by varying the ratio between the A and B solutions.k′_(rel)=(t_(derivative)−t₀)/(t_(human)−t₀). k′_(rel) found for a numberof insulin derivatives according to the invention are given in Table 1.

TABLE 1 Insulin derivative k{acute over ( )}_(rel)N^(εB29)—(N—HOOC(CH₂)₁₄CO-γ-Glu) des(B30) human insulin 0.87N^(εB29)—(N—HOOC(CH₂)₁₆CO-γ-Glu) des(B30) human insulin 1.15N^(εB29)—(N—HOOC(CH₂)₈CO-γ-Glu) des(B30) human insulin 0.45N^(εB29)—(N—HOOC(CH₂)₁₆CO-γ-Glu-N-(γ-Glu) des(B30) human insulin 1.17N^(εB29)—(N-(Asp-OC(CH₂)₁₆CO)-γ-Glu) des(B30) human insulin 0.70N^(εB29)—(N-(Glu-OC(CH₂)₁₄CO-γ-Glu) des(B30) human insulin 0.33N^(εB29)—(N-(Glu-OC(CH₂)₁₄CO—) des(B30) human insulin 1.17N^(εB29)—(N—HOOC(CH₂)₁₆CO-α-Glu)-N-(β-Asp) des(B30) human insulin 1.11N^(εB29)—(N-(Gly-OC(CH₂)₁₃CO-γ-Glu) des(B30) human insulin 0.58N^(εB29)—(N-(Sar-OC(CH₂)₁₃CO-γ-Glu) des(B30) human insulin 0.63N^(εB29)—(N—HOOC(CH₂)₁₆CO-α-Glu)-N-(AspAsp) des(B30) human insulin 1.07N^(εB29)—(N-(Gly-OC(CH₂)₁₄CO-γ-Glu) des(B30) human insulin 0.88N^(εB29)—(N—HOOC(CH₂)₁₅CO-γ-L-Glu) des(B30) human insulin 1.13N^(εB29)—(N—HOOC(CH₂)₁₄CO-β-L-Asp) des(B30) human insulin 0.69N^(εB29)—(N—HOOC(CH₂)₁₃CO-β-L-Glu) des(B30) human insulin 0.54N^(εB29)—(N—HOOC(CH₂)₁₃CO-β-L-Asp) des(B30) human insulin 0.47N^(εB29)—(N—HOOC(CH₂)₁₆CO-δ-L-Aad) des(B30) human insulin 0.84N^(εB29)—(N—HOOC(CH₂)₁₆CO-γ-D-Glu) des(B30) human insulin 1.4N^(εB29)—(N—HOOC(CH₂)₁₅CO-β-L-Asp) des(B30) human insulin 1.09N^(εB29)—(N—HOOC(CH₂)₁₆CO-α-L-Asp) des(B30) human insulin 1.49N^(εB29)—(N—HOOC(CH₂)₁₆CO-α-L-Glu) des(B30) human insulin 1.51N^(εB29)—(N—(HOOC(CH₂)₁₄CO-ε-L-LysCO—) des(B30) human insulin 0.90N^(εB29)—(N—HOOC(CH₂)₁₆CO-β-L-Asp) des(B30) human insulin 1.54N^(εB29)—(N-(Gly-OC(CH₂)₁₆CO-γ-L-Glu) des(B30) human insulin 1.57N^(εB29)—[N—(HOOC(CH₂)₁₆CO)—N-(carboxymethyl)-β-Ala] des(B30) humaninsulin 1.13 N^(εB29)—[N^(α)—(HOOC(CH₂)₁₁)NHCO(CH₂)₃CO)-γ-L-Glu]des(B30) human insulin 0.42

PHARMACEUTICAL COMPOSITIONS

Pharmaceutical compositions containing an insulin derivative accordingto the present invention may be administered parenterally to patients inneed of such a treatment. Parenteral administration may be performed bysubcutaneous, intramuscular or intravenous injection by means of asyringe, optionally a pen-like syringe. Alternatively, parenteraladministration can be performed by means of an infusion pump. Furtheroptions are to administer the insulin nasally or pulmonally, preferablyin compositions, powders or liquids, specifically designed for thepurpose.

Injectable compositions of the insulin derivatives of the invention canbe prepared using the conventional techniques of the pharmaceuticalindustry which involve dissolving and mixing the ingredients asappropriate to give the desired end product. Thus, according to oneprocedure, an insulin derivative according to the invention is dissolvedin an amount of water which is somewhat less than the final volume ofthe composition to be prepared. An isotonic agent, a preservative and abuffer is added as required and the pH value of the solution isadjusted—if necessary—using an acid, e.g. hydrochloric acid, or a base,e.g. aqueous sodium hydroxide as needed. Finally, the volume of thesolution is adjusted with water to give the desired concentration of theingredients.

In a further embodiment of the invention the buffer is selected from thegroup consisting of sodium acetate, sodium carbonate, citrate,glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogenphosphate, disodium hydrogen phosphate, sodium phosphate, andtris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate,maleic acid, fumaric acid, tartaric acid, aspartic acid or mixturesthereof. Each one of these specific buffers constitutes an alternativeembodiment of the invention.

In a further embodiment of the invention the formulation furthercomprises a pharmaceutically acceptable preservative which may beselected from the group consisting of phenol, o-cresol, m-cresol,p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate,2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzylalcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid,imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethylp-hydroxybenzoate, benzethonium chloride, chlorphenesine(3p-chlorphenoxypropane-1,2-diol) or mixtures thereof. In a furtherembodiment of the invention the preservative is present in aconcentration from 0.1 mg/ml to 20 mg/ml. In a further embodiment of theinvention the preservative is present in a concentration from 0.1 mg/mlto 5 mg/ml. In a further embodiment of the invention the preservative ispresent in a concentration from 5 mg/ml to 10 mg/ml. In a furtherembodiment of the invention the preservative is present in aconcentration from 10 mg/ml to 20 mg/ml. Each one of these specificpreservatives constitutes an alternative embodiment of the invention.The use of a preservative in pharmaceutical compositions is well-knownto the skilled person. For convenience reference is made to Remington:The Science and Practice of Pharmacy, 19^(th) edition, 1995.

In a further embodiment of the invention the formulation furthercomprises an isotonic agent which may be selected from the groupconsisting of a salt (e.g. sodium chloride), a sugar or sugar alcohol,an amino acid (e.g. L-glycine, L-histidine, arginine, lysine,isoleucine, aspartic acid, tryptophan, threonine), an alditol (e.g.glycerol (glycerine), 1,2-propanediol (propyleneglycol),1,3-propanediol, 1,3-butanediol) polyethyleneglycol (e.g. PEG400), ormixtures thereof. Any sugar such as mono-, di-, or polysaccharides, orwater-soluble glucans, including for example fructose, glucose, mannose,sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran,pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch andcarboxymethylcellulose-Na may be used. In one embodiment the sugaradditive is sucrose. Sugar alcohol is defined as a C4-C8 hydrocarbonhaving at least one —OH group and includes, for example, mannitol,sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. In oneembodiment the sugar alcohol additive is mannitol. The sugars or sugaralcohols mentioned above may be used individually or in combination.There is no fixed limit to the amount used, as long as the sugar orsugar alcohol is soluble in the liquid preparation and does notadversely effect the stabilizing effects achieved using the methods ofthe invention. In one embodiment, the sugar or sugar alcoholconcentration is between about 1 mg/ml and about 150 mg/ml. In a furtherembodiment of the invention the isotonic agent is present in aconcentration from 1 mg/ml to 50 mg/ml. In a further embodiment of theinvention the isotonic agent is present in a concentration from 1 mg/mlto 7 mg/ml. In a further embodiment of the invention the isotonic agentis present in a concentration from 8 mg/ml to 24 mg/ml. In a furtherembodiment of the invention the isotonic agent is present in aconcentration from 25 mg/ml to 50 mg/ml. Each one of these specificisotonic agents constitutes an alternative embodiment of the invention.The use of an isotonic agent in pharmaceutical compositions iswell-known to the skilled person. For convenience reference is made toRemington: The Science and Practice of Pharmacy, 19^(th) edition, 1995.

Typical isotonic agents are sodium chloride, mannitol, dimethyl sulfoneand glycerol and typical preservatives are phenol, m-cresol, methylp-hydroxybenzoate and benzyl alcohol.

Examples of suitable buffers are sodium acetate, glycylglycine, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and sodiumphosphate.

A composition for nasal administration of an insulin derivativeaccording to the present invention may, for example, be prepared asdescribed in European Patent No. 272097 (to Novo Nordisk A/S).

Compositions containing insulins of this invention can be used in thetreatment of states which are sensitive to insulin. Thus, they can beused in the treatment of type 1 diabetes, type 2 diabetes andhyperglycaemia for example as sometimes seen in seriously injuredpersons and persons who have undergone major surgery. The optimal doselevel for any patient will depend on a variety of factors including theefficacy of the specific insulin derivative employed, the age, bodyweight, physical activity, and diet of the patient, on a possiblecombination with other drugs, and on the severity of the state to betreated. It is recommended that the daily dosage of the insulinderivative of this invention be determined for each individual patientby those skilled in the art in a similar way as for known insulincompositions.

Where expedient, the insulin derivatives of this invention may be usedin mixture with other types of insulin, e.g. insulin analogues with amore rapid onset of action. Examples of such insulin analogues aredescribed e.g. in the European patent applications having thepublication Nos. EP 214826 (Novo Nordisk A/S), EP 375437 (Novo NordiskA/S) and EP 383472 (Eli Lilly & Co.).

The present invention is further illustrated by the following exampleswhich, however, are not to be construed as limiting the scope ofprotection.

Definitions

By “insulin analogue” as used herein is meant a polypeptide which has amolecular structure which formally can be derived from the structure ofa naturally occurring insulin, for example that of human insulin, bydeleting and/or exchanging at least one amino acid residue occurring inthe naturally occurring insulin and/or adding at least one amino acidresidue. The added and/or exchanged amino acid residues can either becodable amino acid residues or other naturally occurring residues orpurely synthetic amino acid residues The insulin analogues may be suchwherein position 28 of the B chain may be modified from the natural Proresidue to one of Asp, Lys, or Ile. In another embodiment Lys atposition B29 is modified to Pro. In one embodiment B30 may be Lys andthen B29 can be any codable amino acid except Cys, Met, Arg and Lys.

Also, Asn at position A21 may be modified to Ala, Gln, Glu, Gly, His,Ile, Leu, Met, Ser, Thr, Trp, Tyr or Val, in particular to Gly, Ala,Ser, or Thr and preferably to Gly. Furthermore, Asn at position B3 maybe modified to Lys or Asp. Further examples of insulin analogues aredes(B30) human insulin; des(B30) human insulin analogues; insulinanalogues wherein PheB1 has been deleted; insulin analogues wherein theA-chain and/or the B-chain have an N-terminal extension and insulinanalogues wherein the A-chain and/or the B-chain have a C-terminalextension. Thus one or two Arg may be added to position B1.

By “insulin derivative” as used herein is meant a naturally occurringinsulin or an insulin analogue which has been chemically modified, e.g.by introducing a side chain in one or more positions of the insulinbackbone or by oxidizing or reducing groups of the amino acid residuesin the insulin or by converting a free carboxylic group to an estergroup or acylating a free amino group or a hydroxy group.

The expression “a codable amino acid” or “a codable amino acid residue”is used to indicate an amino acid or amino acid residue which can becoded for by a triplet (“codon”) of nucleotides.

hGlu is homoglutamic acid.

α-Asp is the L-form of —HNCH(CO—)CH₂COOH.

β-Asp is the L-form of —HNCH(COOH)CH₂CO—.

α-Glu is the L-form of —HNCH(CO—)CH₂CH₂COOH.

γ-Glu is the L-form of —HNCH(COOH)CH₂CH₂CO—.

α-hGlu is the L-form of —HNCH(CO—)CH₂CH₂CH₂COOH.

δ-hGlu is the L-form of —HNCH(COOH)CH₂CH₂CH₂CO—.

β-Ala is —NH—CH₂—CH₂—COOH.

Sar is sarcosine (N-methylglycine).

The expression “an amino acid residue having a carboxylic acid group inthe side chain” designates amino acid residues like Asp, Glu and hGlu.The amino acids can be in either the L- or D-configuration. If nothingis specified it is understood that the amino acid residue is in the Lconfiguration.

The expression “an amino acid residue having a neutral side chain”designates amino acid residues like Gly, Ala, Val, Leu, Ile, Phe, Pro,Ser, Thr, Cys, Met, Tyr, Asn and Gln.

When an insulin derivative according to the invention is stated to be“soluble at physiological pH values” it means that the insulinderivative can be used for preparing injectable insulin compositionsthat are fully dissolved at physiological pH values. Such favourablesolubility may either be due to the inherent properties of the insulinderivative alone or a result of a favourable interaction between theinsulin derivative and one or more ingredients contained in the vehicle.

The following abbreviations have been used in the specification andexamples:

Aad: Alpha-amino-adipic acid (homoglutamic acid)

Bzl=Bn: benzyl

DIEA: N,N-diisopropylethylamine

DMF: N,N-dimethylformamide

IDA: Iminodiacetic acid

Sar: Sarcosine (N-methyl-glycine)

tBu: tert-butyl

TSTU: O—(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate

THF: Tetrahydrofuran

EtOAc: Ethyl acetate

DIPEA: Diisopropylethylamine

HOAt: 1-Hydroxy-7-azabenzotriazole

TEA: triethyl amine

Su: succinimidyl=2,5-dioxo-pyrrolidin-1-yl

TFA: trifluoracetic acid

DCM: dichloromethane

DMSO: dimethyl sulphoxide

TLC: Thin Layer Chromatography

RT: room temperature

EXAMPLES Example 1

Synthesis of N^(εB29)—(N^(α)-(HOOC(CH₂)₁₄CO)-γ-Glu) des(B30) HumanInsulin

200 mg of des(B30) human insulin was dissolved in 10 ml of 50 mM Na2CO3(pH 10.2) contained in a tube which was placed in a water bath at 15° C.Methyl hexadecandioyl-Glu(OSu)-OMe (37.90 mg, prepared as describedbelow), was dissolved in 10 ml of acetonitrile and subsequently added tothe insulin solution. The reaction was stopped after 30 min by additionof 3.8 ml of 0.2 M ethanolamine adjusted to pH 9.0 with dilute HCl. Theyield of the reaction was 37% as determined by RP-HPLC. The productprecipitated after addition of 2.5 volumes of water and adjustment of pHto 5.5. The precipitate was then dissolved in 10 ml of water at pH 8 andplaced in ice. To this solution was added 10 ml of ice cold 0.2 M NaOHfor saponification and the mixture was incubated for 40 min with icecooling and then adjusted to pH 5.5 to precipitate the product. Theprecipitate was isolated and dissolved in 5 ml of A-buffer (see below)and diluted with 33 ml of 42.5% w/w aqueous ethanol divided in three andsubjected to anion exchange chromatography employing a Resource™ 6 mlanion exchange column eluted with a buffer system consisting ofA-buffer: Tris 0.24% w/w, NH4Ac 0.25%, 42% ethanol w/w, pH 7.5 andB-buffer: Tris 0.24% w/w, NH4Ac 1.25%, 42% ethanol w/w pH 7.5. Thesample was eluted by a flow of 6 ml/min in a gradient from 0 to 100% ofB-buffer in 30 min. The fractions containing the desired compound wereidentified by RP-HPLC. The yield of the desired product was 15.3 mg(purity: 72.9%). The volume of the pooled fractions containing thedesired compound was reduced to 20 ml under vacuum and this solution wasthen subjected to purification by RP-HPLC employing a reversed phaseHPLC column Nucleosil, C4 250/10 mm, 10 μm, 300 Å. The buffer systemconsisted of A-buffer: 10 mM Tris, 15 mM (NH4)2SO4, 10% ethanol, pH 7.3and B-buffer: 70% vol/vol ethanol.

The product was eluted with a gradient of from 10% to 60% of B-buffer in120 min at a flow of 2 ml/min. The appropriate fractions were pooled andthe compound was precipitated and lyophilized. The yield was 7.7 mg(purity: 99.4%).

Molecular weight, found by mass spectroscopy: 6097.2, calculated 6104.1.B-terminal peptide containing the side chain was obtained afterdigestion with staphylococcus aureus protease. Molecular weight, foundby mass spectroscopy: 1413.1, calculated: 1413.5.

Preparation of Methyl Hexadecandioyl-Glu(OSu)-OMe

Dimethyl hexadecandioate was saponified in MeOH using 1.0 equivalent ofNaOH, and the mono-methyl ester was isolated upon HCl acidification byrecrystallisation from heptane.

Mono-methyl hexadecandioate (275 mg, 0.91 mmol) was dissolved in THF (3ml) and treated with succinimidyl tetramethyluroniumtetrafluoroborate(331 mg, 1.1 mmol) and N,N-diisopropylethylamine (188 μL, 1.1 mmol), andthe mixture was stirred for 20 hours. The solvent was removed in vacuo,and the residue was dissolved in ethyl acetate and washed with 0.1 M HCl(twice) and water. The organic phase was dried over MgSO4, filtered andevaporated in vacuo to give 350 mg (96%) of methyl succinimidylhexadecandioate.

1H-NMR (CDCl3) □: 3.66 (s, 3H), 2.83 (s, 4H), 2.60 (t, 2H), 2.30 (t,2H), 1.74 (p, 2H), 1.62 (p, 2H), 1.40 (m, 2H), 1.35-1.22 (m, 18 H).

Methyl succinimidyl hexadecandioate (240 mg, 0.58 mmol) indimethylformamide (5 ml) was treated with GluOMe (93 mg, 0.58 mmol) andN,N-diisopropylethylamine (200 μL, 1.16 mmol). The mixture was stirredfor 20 hours and then evaporated in vacuo. The residue was redissolvedin ethyl acetate. Washing with 0.1 M HCl and water, followed by drying(MgSO4) and evaporation in vacuo, gave 226 mg (88%) of methylhexadecandioyl-Glu-OMe.

1H-NMR □: 6.22 (d, 1H), 4.65 (m, 1H), 3.76 (s, 3H), 3.66 (s, 3H), 2.42(t, 2H), 2.29 (m, 4H), 2.22 (t, 2H), 1.97 (m, 2H), 1.62 (m, 4H),1.35-1.22 (m, 20 H).

Methyl hexadecandioyl-Glu-OMe (200 mg, 0.45 mmol) was dissolved indichloromethane (4 ml), cooled with an ice-bath and treated withdicyclohexylcarbodiimide (93 mg, 0.45 mmol) and N-hydroxysuccinimide (52mg, 0.45 mmol). The mixture was stirred for 20 hours, filtered andevaporated in vacuo, to give 243 mg (100%) of the desired intermediate.

1H-NMR (CDCl3) □: 6.34 (d, 1H), 4.67 (m, 1H), 3.73 (s, 3H), 3.64 (s,3H), 2.81 (s, 4H), 2.66 (m, 2H), 2.27 (m, 4H), 2.20 (t, 2H), 1.89 (m,1H), 1.70 (m, 1H), 1.58 (m, 4H), 1.291.20 (m, 20 H).

Example 2

Synthesis of NεB29—(Nα-(HOOC(CH2)16CO)-γ-Glu) des(B30) Human Insulin

300 mg of des(B30) human insulin was acylated, purified and isolated asdescribed in Example 1, except that in the present example methyloctadecandioyl-Glu(OSu)-OMe (prepared as described below) was used asacylating agent instead of the methyl hexadecandioyl-Glu(OSu)-OMe usedin Example 1. 25.5 mg of the title compound was obtained (purity:97.4%). Molecular weight, found by mass spectroscopy: 6136.6,calculated: 6132. B-terminal peptide containing the ligand was obtainedafter digestion by staphylococcus aureus protease. Molecular weight,found by mass spectroscopy: 1439.1, Calculated: 1442.5.

Preparation of Methyl Octadecandioyl-Glu(OSu)-OMe

This compound was prepared from dimethyl octadecandioate in analogy withthe hexadecandioyl derivative described in Example 1.

1H-NMR (CDCl3) □: 6.20 (d, 1H), 4.70 (m, 1H), 3.78 (s, 3H), 3.67 (s,3H), 2.84 (s, 4H), 2.70 (m, 2H), 2.30 (m, 4H), 2.22 (t, 2H), 1.93 (m,1H), 1.70 (m, 1H), 1.62 (m, 4H), 1.33-1.23 (m, 24 H).

Example 3

Synthesis of N^(εB29)—(N^(α)-(HOOC(CH₂)₁₆CO)-γ-Glu—N-(γ-Glu)) des(B30)Human Insulin

In a similar way as described in Example 1, 300 mg of des(B30) humaninsulin was acylated with methyl octadecandioyl-Glu(Glu(OSu)-OMe)-OMe.Purification by anion exchange was performed as described above.However, prolonged elution with 100% B-buffer was conducted in order toelute the desired product. The fractions containing the desired productwere identified by RP-HPLC and 47.5 mg was obtained at a purity of 55%.The volume of the fractions containing the desired product was reducedin vacuo and the resulting solution was subjected to purification byRP-HPLC employing a reversed phase HPLC Jupiter column, C4 250/10 mm, 10μm, 300 Å from Phenomerex. The buffer system consisted of A-buffer: 0.1%TFA, 10% vol/vol ethanol and B-buffer: 80% vol/vol ethanol. The samplewas eluted by a gradient from 40% to 60% B-buffer at 40° C. for 120 minat a flow of 2 ml/min. The appropriate fractions were pooled andlyophilized and 31.1 mg of the title compound was obtained (purity:94%).

Molecular weight of the title compound found by mass spectroscopy:6259.65, calculated: 6261.2. The molecular weight (by mass spectroscopy)of the B-terminal peptide containing the side chain, obtained afterdigestion with staphylococcus aureus protease was found to be 1569.88,calculated: 1569.88.

Preparation of Methyl Octadecandioyl-Glu(Glu(OSu)-OMe)-OMe

Methyl octadecandioyl-Glu(OSu)-OMe (prepared as described in Example 2,200 mg, 0.35 mmol) in dimethylformamide (5 ml) was treated with GluOMe(62 mg, 0.39 mmol) and N,N-diisopropylethylamine (90 μL, 53 mmol), andthe mixture was stirred for 20 hours. The solvent was removed in vacuo,and the residue was dissolved in ethyl acetate and washed twice with 0.2M HCl, water and brine. Drying over MgSO₄ and evaporation gave methyloctadecandioyl-Glu(Glu-OMe)-OMe, 180 mg (83%).

Methyl octadecandioyl-Glu(Glu-OMe)-OMe (180 mg, 0.29 mmol) was dissolvedin THF (9 ml) and treated with succinimidyltetramethyluroniumtetrafluoroborate (106 mg, 0.35 mmol) andN,N-diisopropylethylamine (60 μL, 0.35 mmol). The mixture was stirredovernight, evaporated, redissolved in ethyl acetate, and washed with2×0.1 M HCl and water. Drying over MgSO₄ and evaporation gave 190 mg(93%) of the desired intermediate.

¹H-NMR (CDCl₃) δ: 6.73 (d, 1H), 6.43 (d, 1H), 4.69 (m, 1H), 4.56 (m,1H), 3.77 (s, 3H), 3.74 (s, 3H), 3.66 (s, 3H), 2.85 (s, 4H), 2.72 (m,2H), 2.41-2.12 (m, 8H), 2.95 (m, 2H), 1.72-1.56 (m, 6H), 1.35-1.22 (m,22 H).

Example 4

Synthesis of N^(εB29)—(N^(α)-(HOOC(CH₂)₁₄CO)-γ-L-Glu) des(B30) HumanInsulin

Des(B30) human insulin (500 mg, 0.088 mmol) was dissolved in 100 mMNa₂CO₃ (5 ml, pH 10.2) at room temperature. Tert-butylhexadecandioyl-Glu(OSu)-OtBu (66 mg, 0.105 mmol, prepared as describedbelow), was dissolved in acetonitrile (5 ml) and subsequently added tothe insulin solution. After 30 mins, 0.2 M methylamine (0.5 ml) wasadded. pH was adjusted by HCl to 5.5, and the isoelectric precipitatewas collected by centrifugation and dried in vacuo to give 525 mg. Thecoupling yield was 78% (RP-HPLC, C4 column; Buffer A: 10% MeCN in 0.1%TFA-water, Buffer B: 80% MeCN in 0.1% TFA-water; gradient 20% to 90% Bin 16 minutes). The protected product was dissolved in TFA (10 ml), left30 mins, and evaporated in vacuo. The crude product was dissolved inwater and lyophilized (610 mg). 0454 was purified by RP-HPLC onC4-column, buffer A: 20% EtOH+0.1% TFA, buffer B: 80% EtOH+0.1% TFA;gradient 15-60% B, followed by HPLC on C4-column, buffer A: 10 mMTris+15 mM ammonium sulphate in 20% EtOH, pH 7.3, buffer B: 80% EtOH,gradient 15-60% B. The collected fractions were desalted on Sep-Pak with70% acetonitrile+0.1% TFA, neutralized by addition of ammonia andfreeze-dried. The unoptimized yield was 64 mg, 12%. The purity asevaluated by HPLC was 99.2%. LCMS 6102.9, C₂₇₄H₄₁₁N₆₅O₈₁S₆ requires6104.1. B-terminal peptide containing the side chain(RGFFYTPK(N^(ε)-(N^(α)-(HOOC(CH₂)₁₄CO)-γ-L-Glu) was obtained afterdigestion with staphylococcus aureus protease. MALDI-MS: 1413.1,calculated: 1412.7.

Preparation of Tert-Butyl Hexadecandioyl-L-Glu(OSu)-OtBu

Hexadecadioic acid (40.0 g, 140 mmol) was suspended in toluene (250 ml)and the mixture was heated to reflux. N,N-dimethylformamidedi-tert-butyl acetal (76.3 g, 375 mmol) was added drop-wise over 4hours. The mixture was refluxed overnight. The solvent was removed invacuo at 50° C., and the crude material was suspended in DCM/AcOEt (500ml, 1:1) and stirred for 15 mins. The solids were collected byfiltration and triturated with DCM (200 ml). The filtrated wereevaporated in vacuo to give crude mono-tert-butyl hexadecandioate, 30grams. This material was suspended in DCM (50 ml), cooled with ice for10 mins, and filtered. The solvent was removed in vacuo to leave 25 gramcrude mono-tert-butyl hexadecandioate, which was recrystallized fromheptane (200 ml) to give mono-tert-butyl hexadecandioate, 15.9 g (33%).Alternatively to recrystallization, the mono-ester can be purified bysilica chromatography in AcOEt/heptane.

¹H-NMR (CDCl₃) δ: 2.35 (t, 2H), 2.20 (t, 2H), 1.65-1.55 (m, 4H), 1.44(s, 9H), 1.34-1.20 (m, 20 H).

The mono tert-butyl ester (2 g, 5.8 mmol) was dissolved in THF (20 ml)and treated with TSTU (2.1 g, 7.0 mmol) and DIEA (1.2 ml, 7.0 mmol) andstirred overnight. The mixture was filtered, and the filtrate wasevaporated in vacuo. The residue was dissolved in AcOEt and washed twicewith cold 0.1 M HCl and water. Drying over MgSO₄ and evaporation invacuo gave succinimidyl tert-butyl hexadecandioate, 2.02 g (79%).

¹H-NMR (CDCl₃) δ: 2.84 (s, 4H), 2.60 (t, 2H), 2.20 (t, 2H), 1.74 (p,2H), 1.56 (m, 2H), 1.44 (s, 9H), 1.40 (m, 2H), 1.301.20 (m, 18H).

Succinimidyl tert-butyl hexadecandioate (1 g, 2.27 mmol) was dissolvedDMF (15 ml) and treated with L-Glu-OtBu (0.51 g, 2.5 mmol) and DIEA(0.58 ml, 3.41 mmol) and the mixture was stirred overnight. The solventwas evaporated in vacuo, and the crude product was dissolved in AcOEt,and washed twice with 0.2M HCl, with water and brine. Drying over MgSO₄and evaporation in vacuo gave tert-butyl hexadecandioyl-L-Glu-OtBu, 1.2g (100%).

¹H-NMR (CDCl₃) δ: 6.25 (d, 1H), 4.53 (m, 1H), 2.42 (m, 2H), 2.21 (m,4H), 1.92 (m, 1H), 1.58 (m, 4H), 1.47 (s, 9H), 1.43 (s, 9H), 1.43-1.22(m, 18H).

Tert-butyl hexadecandioyl-L-Glu-OtBu (1.2 g, 2.27 mmol) was dissolved inTHF (15 ml) and treated with TSTU (0.82 g, 2.72 mmol) and DIEA (0.47 ml,2.72 mmol) and stirred overnight. The mixture was filtered, and thefiltrate was evaporated in vacuo. The residue was dissolved in AcOEt andwashed twice with cold 0.1 M HCl and water. Drying over MgSO₄ andevaporation in vacuo gave tert-butyl hexadecandioyl-L-Glu(OSu)-OtBu,1.30 g (92%).

¹H—NMR (CDCl₃) δ: 6.17 (d, 1H), 4.60 (m, 1H), 2.84 (s, 4H), 2.72 (m,1H), 2.64 (m, 1H), 2.32 (m, 1H), 2.20 (m, 4H), 2.08 (m, 1H), 1.6 (m,4H), 1.47 (s, 9H), 1.43 (s, 9H), 1.33-1.21 (m, 20 H).

Example 5

Synthesis of N^(εB29)—(N^(α)-(HOOC(CH₂)₁₆CO)-γ-L-Glu) des(B30) HumanInsulin

DesB30 insulin (50 mg, 9 μmol) was dissolved in 0.1 M aqeous Na₂CO₃(0.65 ml), pH 10.5. Octadecandioyl-L-Glu(OSu) (50.1 mg, 9.9 μmol,prepared as described below) was dissolved in acetonitrile (0.65 ml) andadded to the insulin solution; pH was 10.3. After 30 mins, 0.2 Mmethylamine (50 μl) was added. pH was adjusted by HCl to 5.5, and theisoelectric precipitate was collected by centrifugation and dried invacuo. HPLC showed the crude coupling yield to be 52% (not optimized);C4 column; Buffer A: 10% MeCN in 0.1% TFA-water, Buffer B: 80% MeCN in0.1% TFA-water; gradient 20% to 90% B in 16 minutes). LCMS 6133.2,C₂₇₆H₄₁₅N₆₅O₈₁S₆ requires 6132.2.

Preparation of Octadecandioyl-L-Glu(OSu)

Octadecanedioic acid (2.5 g, 8.0 mmol) was suspended in DCM (60 mL),treated with triethylamine (1.16 mL, 8.3 mmol) and ice-cooled.Benzylchloroformate (1.14 mL) was added drop-wise under nitrogen and themixture was stirred for 10 min, when DMAP (0,097 g, 0.80 mmol) wasadded. After stirring for 20 min at 4° C. (TLC, 1:1 AcOEt:heptane), thereaction was evaporated to dryness. The crude material (3.9 g) wasdissolved in DCM (60 ml), treated with silica (15 g) and evaporated. Thesilica was loaded on a silica column (175 g), and the product was elutedwith AcOEt/heptane 1:7 to 1:1. Evaporation of the desired fractions gavemono-benzyl octadecandioate (1.15 g, 36%).

¹H-NMR (CDCl₃) δ: 7.35 (m, 5H), 5.11 (s, 2H), 2.35 (t, 4H), 1.63 (t,4H), 1.30-1.22 (m, 24).

Mono-benzyl octadecandioate was dissolved in DMF (3.5 mL) and THF (7 mL)and cooled with ice bath. DIEA (0.103 mL) and TSTU were added and themixture was stirred 1 h at ice bath and at RT overnight. The solvent wasevaporated in vacuo and the residue was dissolved in AcOEt and washedtwice with 0.2 N HCl, saturated NaHCO₃, dried, filtered and evaporatedto dryness to give succinimidyl mono-benzyl octadecandioate_(0.25 g,100%).

¹H-NMR (CDCl₃) δ: 7.35 (m, 5H), 5.11 (s, 2H), 2.83 (s, 4H), 2.60 (t,2H), 2.35 (t, 2H), 1.80-1.60 (m, 4H), 1.40-1.20 (m, 24).

Succinimidyl mono-benzyl octadecandioate_(95 mg, 0.19 mmol) wasdissolved DMF (1.5 ml) and treated with L-Glu-OBzl (49 mg, 0.21 mmol)and DIEA (50 □I, 0.28 mmol) and the mixture was stirred overnight. Thesolvent was evaporated in vacuo, and the crude product was dissolved inAcOEt, and washed twice with 0.2M HCl, with water and brine. Drying overMgSO₄ and evaporation in vacuo gave BzlO-octadecandioyl-L-Glu-OBzl, 114mg (97%).

¹H-NMR (CDCl₃) δ: 7.35 (m, 5H), 6.22 (d, 2H), 5.17 (s, 2H), 5.11 (s,2H), 4.71 (m, 1H), 2.37 (m, 4H) , 2.22 (m, 3H), 1.98 (m, 1H), 1.63 (m,4H), 1.31-1.20 (m, 24H).

BzlO-octadecandioyl-L-Glu-OBzl (110 g, 0.18 mmol) was dissolved in THF(2 ml) and treated with TSTU (64 mg, 0.21 mmol) and DIEA (36 μl, 0.21mmol) and stirred overnight. The mixture was filtered, and the filtratewas evaporated in vacuo. The residue was dissolved in AcOEt and washedtwice with cold 0.1 M HCl and water. Drying over MgSO₄ and evaporationin vacuo gave BzlO-octadecandioyl-L-Glu(OSu)-OBzl, 119 g (94%).

¹H-NMR (CDCl₃) δ: 7.36 (m, 5H), 6.40 (d, 2H), 5.19 (s, 2H), 5.11 (s,2H), 4.75 (m, 1H), 2.82 (s, 4H), 2.68 (m, 1H), 2.59 (m, 1H), 2.35 (t,2H), 2.19 (t, 2H), 1.62 (m, 4H), 1.32-1.21 (m, 24H).

BzlO-octadecandioyl-L-Glu(OSu)-OBzl (59 mg, 0.082 mmol) was dissolved inacetone/0.1% TFA (1 ml). Pd/C was added (20 mg). The flask was evacuatedand filled with N₂ several times, and a H₂ filled balloon was connected.The mixture was stirred at RT 3 h, and then filtered through celite.Precipitation from heptane and evaporation of residual solvents gaveoctadecandioyl-L-Glu(OSu) (27 mg, 61%).

¹H-NMR (CDCl₃) δ: 6.32 (d, 1H), 4.70 (m, 1H), 3.70 (m, 1H), 3.06 (m,2H), 2.88 (s, 4H), 2.62 (m, 2H), 2.35 (m, 2H), 2.24 (m, 1H), 1.74 (m,1H), 1.64 (m, 2H), 1.50-1.20 (m, 26 H).

Example 6

Synthesis of N^(εB29)—(N—(L-Asp-OC(CH₂)₁₆CO)-γ-L-Glu) des(B30) HumanInsulin

his compound was prepared in analogy with example 4, via reaction ofL-Asp(OtBu)-OtBu with succinimidyl octadecandioate followed byactivation with TSTU, reaction with L-GluOtBu, activation with TSTU,coupling with Des(B30) human insulin and deprotection by TFA.

LCMS 6247.5, calculated 6247.3.

Example 7

Synthesis of N^(εB29)—(N—(L-Glu-OC(CH₂)₁₄CO-γ-L-Glu) des(B30) HumanInsulin

This compound was prepared in analogy with example 4, via reaction ofL-Glu(OtBu)-OtBu with succinimidyl hexadecandioate followed byactivation with TSTU, reaction with L-GluOtBu, activation with TSTU,coupling with Des(B30) human insulin and deprotection by TFA. LCMS6261.3, calculated 6261.3.

Example 8

Synthesis of N^(εB29)—(N—(L-Glu-OC(CH₂)₁₄CO—) des(B30) Human Insulin

This compound was prepared in analogy with example 4, via reaction ofL-Glu(OtBu)-OtBu with succinimidyl hexadecandioate followed byactivation with TSTU, coupling with Des(B30) human insulin anddeprotection by TFA. LCMS 6130.8, calculated 6132.2.

Example 9

Synthesis of N^(εB29)—(N—HOOC(CH₂)₁₆CO-α-L-Glu)—N—(β-L-Asp) des(B30)Human Insulin

This compound was prepared in analogy with example 4, via reaction oftert-butyl succinimidyl octadecandioate with L-Glu(OtBu) followed byactivation with TSTU, reaction with L-AspOtBu, activation with TSTU,coupling with Des(B30) human insulin and deprotection by TFA. LCMS6246.9, calculated 6247.3.

Example 10

Synthesis of N^(εB29)—(N—HOOC(CH₂)₁₅CO-γ-L-Glu) des(B30) Human Insulin

This compound was prepared in analogy with example 4, via reaction oftert-butyl succinimidyl heptadecandioate (A. C. Cope, U. Axen, E. P.Burrows, J. Weinlich, J. Am. Chem Soc. 1966, 88, 4228) with L-GluOtBufollowed by activation with TSTU, coupling with Des(B30) human insulinand deprotection by TFA. LCMS 6118.3, calculated 6118.1.

Example 11

Synthesis of N^(εB29)—(N—(Gly-OC(CH₂)₁₃CO-γ-L-Glu) des(B30) HumanInsulin

This compound was prepared in analogy with example 4, via reaction ofL-GlyOtBu with succinimidyl pentadecandioate followed by activation withTSTU, reaction with L-GluOtBu, activation with TSTU, coupling withDes(B30) human insulin and deprotection by TFA. LCMS 6147.5, calculated6147.1.

Example 12

Synthesis of N^(εB29)—(N—(L-Sar-OC(CH₂)₁₃CO-γ-L-Glu) des(B30) HumanInsulin

This compound was prepared in analogy with example 4, via reaction ofL-Sar-OtBu with succinimidyl octadecandioate, followed by activationwith TSTU, reaction with L-GluOtBu, activation with TSTU, coupling withDes(B30) human insulin and deprotection by TFA. LCMS 6161.0, calculated6161.1.

Example 13

Synthesis of N^(εB29)—(N—HOOC(CH₂)₁₆CO-α-L-Asp)—N-(β-L-Asp) des(B30)Human Insulin

This compound was prepared in analogy with example 4, via reaction oftert-butyl succinimidyl octadecandioate with L-Asp(OtBu) followed byactivation with TSTU, reaction with L-AspOtBu, activation with TSTU,coupling with Des(B30) human insulin and deprotection by TFA. LCMS6233.8, calculated 6233.2.

Example 14

Synthesis of N^(εB29)—(N—(Gly-OC(CH₂)₁₄CO-γ-L-Glu) des(B30) HumanInsulin

This compound was prepared in analogy with example 4, via reaction ofL-GlyOtBu with succinimidyl hexadecandioate followed by activation withTSTU, reaction with L-GluOtBu, activation with TSTU, coupling withDes(B30) human insulin and deprotection by TFA. LCMS 6160.7, calculated6161.1.

Example15

Synthesis of N^(εB29)—(N—HOOC(CH₂)₁₄CO-β-L-Asp) des(B30) Human Insulin

This compound was prepared in analogy with example 4, via reaction oftert-butyl succinimidyl hexadecandioate with L-AspOtBu followed byactivation with TSTU, coupling with Des(B30) human insulin anddeprotection by TFA. LCMS 6089.8, calculated 6089.8.

Example 16

Synthesis of N^(εB29)—(N—HOOC(CH₂)₁₆CO-β-L-Asp) des(B30) Human Insulin

This compound was prepared in analogy with example 4, via reaction oftert-butyl succinimidyl octadecandioate with L-AspOtBu followed byactivation with TSTU, coupling with Des(B30) human insulin anddeprotection by TFA. LCMS 6117.8, calculated 6118.1.

Example 17

Synthesis of N^(εB29)—(N—(Gly-OC(CH₂)₁₆CO-γ-L-Glu) des(B30) HumanInsulin

This compound was prepared in analogy with example 4, via reaction ofL-GlyOtBu with succinimidyl octadecandioate followed by activation withTSTU, reaction with L-GluOtBu, activation with TSTU, coupling withDes(B30) human insulin and deprotection by TFA. LCMS 6189.2, calculated6189.2.

Example 18

Synthesis of N^(εB29)—(N—(HOOC(CH₂)₁₄CO-ε-L-LysCO—) des(B30) HumanInsulin

This compound was prepared in analogy with example 4, via reaction oftert-butyl succinimidyl hexadecandioate with L-Lys(Z)-OtBu, followed byhydrogenation over Pd/C and in-situ activation by 4-nitrophenylchloroformate, coupling with Des(B30) human insulin and deprotection byTFA. LCMS 6189.2, calculated 6189.2.

Example 19

Synthesis of N^(εB29)—(N—HOOC(CH₂)₁₆CO-α-L-Glu) des(B30) Human Insulin

This compound was prepared in analogy with example 4, via reaction oftert-butyl succinimidyl octadecandioate with L-Glu(OtBu) followed byactivation with TSTU, coupling with Des(B30) human insulin anddeprotection by TFA. LCMS 6132.1, calculated 6132.2.

Example 20

Synthesis of N^(εB29)—(N—HOOC(CH₂)₁₆CO-α-L-Asp) des(B30) Human Insulin

This compound was prepared in analogy with example 4, via reaction oftert-butyl succinimidyl octadecandioate with L-Asp(OtBu) followed byactivation with TSTU, coupling with Des(B30) human insulin anddeprotection by TFA. LCMS 6117.8, calculated 6118.1.

Example 21

Synthesis of N^(εB29)—(N—HOOC(CH₂)₁₅CO-β-L-Asp) des(B30) Human Insulin

This compound was prepared in analogy with example 4, via reaction oftert-butyl succinimidyl heptadecandioate with L-AspOtBu followed byactivation with TSTU, coupling with Des(B30) human insulin anddeprotection by TFA. LCMS 6104.2, calculated 6104.1.

Example 22

Synthesis of N^(εB29)—(N—HOOC(CH₂)₁₆CO-γ-D-Glu) des(B30) Human Insulin

This compound was prepared in analogy with example 4, via reaction oftert-butyl succinimidyl octadecandioate with D-GluOtBu followed byactivation with TSTU, coupling with Des(B30) human insulin anddeprotection by TFA. LCMS 6132.4, calculated 6132.2.

Example 23

Synthesis of N^(εB29)—(N—HOOC(CH₂)₁₆CO-δ-L-Aad) des(B30) Human Insulin

This compound was prepared in analogy with example 4, via reaction oftert-butyl succinimidyl octadecandioate with L-AadOtBu (prepared fromcommercial L-Aad(OMe) by tert-butylation with AcOtBu/BF₃.OEt₂ andsaponification of the methyl ester), followed by activation with TSTU,coupling with Des(B30) human insulin and deprotection by TFA. LCMS6116.9, calculated 6118.1.

Example 24

Synthesis of N^(εB29)—(N—HOOC(CH₂)₁₃CO-β-L-Asp) des(B30) Human Insulin

This compound was prepared in analogy with example 4, via reaction oftert-butyl succinimidyl pentadecandioate with L-AspOtBu followed byactivation with TSTU, coupling with Des(B30) human insulin anddeprotection by TFA. LCMS 6074.7, calculated 6076.1.

Example 25

Synthesis of N^(εB29)—(N—HOOC(CH₂)₁₃CO-β-L-Glu) des(B30) Human Insulin

This compound was prepared in analogy with example 4, via reaction oftert-butyl succinimidyl pentadecandioate with L-GluOtBu followed byactivation with TSTU, coupling with Des(B30) human insulin anddeprotection by TFA. MALDI-MS 6080.6, calculated 6076.1.

Example 26

Synthesis of N^(εB29)—(N—HOOC(CH₂)₁₄CO-β-D-Asp) des(B30) Human Insulin

This compound was prepared in analogy with example 4, via reaction oftert-butyl succinimidyl hexadecandioate with D-AspOtBu followed byactivation with TSTU, coupling with Des(B30) human insulin anddeprotection by TFA. LCMS 6089.1, calculated 6090.1.

Example 27

Synthesis of N^(εB29)—(N—HOOC(CH₂)₁₆CO-β-D-Asp) des(B30) Human Insulin

This compound was prepared in analogy with example 4, via reaction oftert-butyl succinimidyl octadecandioate with D-AspOtBu followed byactivation with TSTU, coupling with Des(B30) human insulin anddeprotection by TFA. LCMS 6117.1, calculated 6118.1.

Example 28

Synthesis of N^(εB29)—(N—HOOC(CH₂)₁₄CO—IDA) des(B30) Human Insulin

This compound was prepared in analogy with example 4, via reaction oftert-butyl 3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl hexadecandioatewith iminodiacetic acid followed by activation with TSTU, coupling withDes(B30) human insulin and deprotection by TFA. LCMS 6089.1, calculated6090.1.

Example 29

Synthesis of N^(εB29)—[N—(HOOC(CH₂)₁₆CO)—N-(carboxymethyl)-β-Ala]des(B30) Human Insulin

A1N, B1N-diBoc DesB30 Human insulin (Kurtzhals P; Havelund S; JonassenI; Kiehr B; Larsen U D; Ribel U; Markussen J Biochemical Journal, 1995,312, 725-731) (186 mg, 0.031 mmol) was dissolved in DMSO (1.8 ml). Asolution of tert-butyloctadecandioyl-N-(tert-butoxycarbonylmethyl)-β-Ala-OSu (27 mg, 0.04mmol) in THF (1.8 ml) and triethylamine (0.045 ml, 0.31 mmol) was added(pH was 10). After slowly stirring at room temperature for 45 min thereaction was quenched with 0.2M methylamine in THF (0,20 ml). Water (5ml) was added and pH was adjusted to 5.5 with 1N HCl. The isoelectricprecipitate was collected by centrifugation and freeze dried to give 150mg. The coupling yield was 74% (LCMS m/z: 2148.9 [(M+3)/3], rt 5.04.)The protected product was dissolved in TFA (2.5 ml) and left for 1 h andevaporated in vacuo. The crude product was purified by RP-HPLC onC4-column, buffer A: 0.1% TFA, buffer B: MeCN+0.1% TFA; gradient 10-70%B. The collected fractions were freeze-dried. The yield was 75 mg, 52%.The purity as evaluated by HPLC was 97.2%.

MALDI-MS: 6132.1, calculated: 6132.2.

Preparation of Tert-ButylOctadecandioyl-N-(tert-butoxycarbonylmethyl)-β-Ala-OSu

Octadecanedioic acid (5.64 g, 17.9 mmol) was dissolved in toluene (80ml) at 115° C. N,N-dimethylformamide di-tert-butylacetal (12.9 ml, 53.8mmol) was added dropwise over 1.5 h. After refluxing for 3 h moreN,N-dimethylformamide di-tert-butylacetal (2.15 ml) was added over 20min. Reflux was continued over night and more N,N-dimethylformamidedi-tert-butylacetal (2.15 ml) was added over 20 min. After stirring foranother 2 h. the reaction mixture was cooled to RT. Water and DCM wasadded. The diacid can was removed by filtration. The filtrate wasconcentrated on silica gel (40 g) and purified on a 1.5 L silicagelcolumn using DCM/MeOH 14:1. Octadecanedioic acid mono-tert-butyl esterwas isolated in 53% yield (3.52 g).

LC-MS: 393 (M+Na), rt 6.40.

¹H-NMR (DMSO-d₆): δ 1.22 (br s, 24H), 1.38 (s, 9H),1.47 (m, 4H), 2.14(t,2H), 2.18 ppm (t, 2H).

To a solution of mono-tert-butyl octadecanedioate (1,00 g, 2.7 mmol) indry THF (8 ml), was added DIPEA (0,555 ml, 3.2 mmol) followed by TSTU(1,00 g, 3.2 mmol). The reaction mixture was stirred under nitrogen for18 h. The solvent had evaporated. AcOEt was added to the residue and theresulting suspension was filtered. The filtrate was washed with cold 0.1M HCl (2×) and water, dried and concentrated to give succinimidyltert-butyl octadecandioate as a white solid.

¹H-NMR (CDCl₃): δ 1.25 (m s, 20H), 1.39 (m, 2H)1.44 (s, 9H),1.58 (m,4H), 1.74 (p, 2H), 2.2 (t,2H), 2.60 (t, 2H), 2,85 ppm (m, 2H).

To a suspension of H-Gly-OtBu (1.00 g, 6.0 mmol) in dry DMF (6 ml) wasadded triethyl amine (0.835 ml, 6.0 mmol). A precipitation of triethylamine hydrochloride was observed. A solution of benzyl acrylate (0.910ml, 6.0 mmol) in DMF (6 ml) was added. The resulting suspension wasstirred at room temperature for 2 days. The precipitate was removed byfiltration and the filtrate was concentrated. The residue was dissolvedin AcOEt and washed with sat. NaHCO₃. The organic layer was dried(MgSO₄), filtered and concentrated to give a clear oil, which waspurified by flash chromatography using AcOEt/heptane 1:3 and 1:1 aseluent. N-ert-butoxycarbonylmethyl)-β-Ala-OBn was isolated in 29% yield(0.505 g).

¹H-NMR (CDCl₃) δ: ppm 1.43 (s, 9H), 2.55 (t, 2H), 2.92 (t, 2H), 3.30 (s,2H), 5.15 (s, 2H), 7.65 (m, 5H).

Succinimidyl tert-butyl octadecandioate (0,15 g, 0.32 mmol) andN-(tert-butoxycarbonylmethyl)-β-Ala-OBn (0.10 g, 0,32 mmol) weredissolved in dry DMF (2.5 ml) and DIEA (0,070 ml, 0.38 mmol) was added.After stirring under nitrogen for 30 min HOAt (0,045 g, 0.32 mmol) wasadded and the mixture turned yellow. Stirring was continued at RT undernitrogen for convenience reasons for 13 days. The reaction mixture wasconcentrated. The residue dissolved in AcOEt, washed with 0.1N HCl (2×)and water, dried (Na₂SO₄) and concentrated to a give tert-butyloctadecandioyl—N-(tert-butoxycarbonylmethyl)-β-Ala-OBn as a white oil.205 mg, Yield 99%.

¹H-NMR (CDCl₃) δ: ppm 1.25 (m, 26H), 1.45 (s, 9H), 1.50 (s, 9H), 1.6 (m,4H), 2.20 (t, 2H), 2.40 (t, 2H), 2.75 (q, 2H), 3.62 (t, 2H), 3.97 (s,2H), 5.20 (s, 2H); 7.35 (m, 5H)

Tert-butyl octadecandioyl-N-(tert-butoxycarbonylmethyl)-β-Ala-OBn (200mg, 0.31 mmol) was dissolved in EtOAc (10 ml) and THF (5 ml). 10% Pd/Cwas added and the mixture was hydrogenated at 1 atm for 16 h. Thereaction mixture was filtered and concentrated to give tert-butyloctadecandioyl—N-(tert-butoxycarbonylmethyl)-β-Ala-OH as a clear oil.Yield 180 mg, 100%.

¹H-NMR (CDCl₃) δ: ppm 1.25 (m, 26H), 1.45 (s, 9H), 1.50 (s, 9H), 1.6 (m,4H), 2.20 (t, 2H), 2.40 (t, 2H), 2.70 (m, 2H), 3.65 (m, 2H), 4.05 (s,2H).

Tert-butyl octadecandioyl-N-(tert-butoxycarbonylmethyl)-β-Ala-OH (0,110g, 0.2 mmol) was dissolved in dry THF (2 ml). DIEA (0,045 ml, 0.24 mmol)and TSTU (0,075 g, 0.24 mmol) was added. The mixture was stirred undernitrogen for 18 h. The reaction mixture was filtered. AcOEt was added tothe filtrate and washed with 0.2 M HCl (2×), brine (1×), dried (Na₂SO₄)and concentrated to give tert-butyloctadecandioyl-N-(tert-butoxycarbonylmethyl)-β-Ala-OSu as a clear sirup.Yield 124 mg, 96%.

¹H-NMR (CDCl₃) δ: ppm 1.25 (m, 26H), 1.40 (s, 9H), 1.57 (s, 9H), 1.6 (m,4H), 2.40 (m, 4H), 2.58 (br s, 4H), 3.0 (t, 2H), 3.7 (t, 2H), 4.03 (s,2H).

Example 30

Synthesis of N^(εB29)—[N—(HOOC(CH₂)₁₆CO)—N-(2-carboxyethyl)-Gly]des(B30) Human Insulin

A1N, B1N-diBoc DesB30 Human insulin (Kurtzhals P; Havelund S; JonassenI; Kiehr B; Larsen U D; Ribel U; Markussen J Biochemical Journal, 1995,312, 725-731) (120 mg, 0.020 mmol) was dissolved in DMSO (1.2 ml). Asolution of tert-butyloctadecandioyl-N-(2-(tert-butoxycarbonyl)ethyl)-Gly-OSu (16 mg, 0.025mmol) in THF (1.2 ml) and triethylamine (0.033 ml, 0.24 mmol) was added(pH was 10). After slowly stirring at room temperature for 3 h and 20min water (4 ml) was added and pH was adjusted to 5.5 with 1N HCl. Theisoelectric precipitate was collected by centrifugation, washed withwater and isolated by centrifugation. The product was freeze dried. Thecrude product was purified by RP-HPLC on C18-column, buffer A: 0.1% TFA,buffer B: MeCN+0.1% TFA; gradient 20-90% B. The collected fractions werefreeze-dried. The unoptimized coupling yield was 15 mg, 11% (MALDI-MS6441, calculated: 6444.5) The protected product was dissolved in TFA (1ml) and left for 1 h and evaporated in vacuo. The crude product waspurified by RP-HPLC on C4-column, buffer A: 0.1% TFA, buffer B:MeCN+0.1% TFA; gradient 10-80% B, and by RP-HPLC on C4-column, buffer A:20% EtOH+0.1% TFA, buffer B: 80% EtOH+0.1% TFA; gradient 15-60% B,followed by HPLC on C4-column, buffer A: 10 mM Tris+15 mM ammoniumsulphate in 20% EtOH, pH 7.3, buffer B: 80% EtOH, gradient 15-60% B. Thecollected fractions were desalted on Sep-Pak with 70% acetonitrile+0.1%TFA, neutralized by addition of ammonia and freeze-dried. Theunoptimized yield was 1.8 mg, 13%. The purity as evaluated by HPLC was96.4%.

MALDI: 6132.1, calculated: 6132.2.

Preparation of Tert-ButylOctadecandioyl-N-(2-(Tert-Butoxycarbonyl)Ethyl)-Gly-OSu

H-Gly-OBn, HCl (3.03 g, 15 mmol) was dissolved in dry DMF (15 ml) andcooled on an ice bath. TEA (2.10, 15 mmol) was added under precipitationof TEA-hydrochloride. The suspension was stirred for 5 min beforet-butyl acrylate (2.20 ml, 15 mmol) was added. The cooling bath wasallowed to reach RT slowly and stirring was continued under nitrogen for2 days. The reaction mixture was filtered and the filtrate wasconcentrated. The residue, still containing DMF, was dissolved in AcOEtand washed with sat aq NaHCO₃ (2×) and water (1×). The organic layer wasfiltered before drying (Na₂SO₄) and concentration to give a yellow oil.Purification by flash chromatography or preparative HPLC gaveN-(2-(tert-butoxycarbonyl)ethyl)-Gly-OBn as a clear oil (0.739 g, 17%).

¹H-NMR (CDCl₃) δ: ppm 1.46 (s, 9H) 2.50-2.61 (m, 2H) 2.82-2.99 (m, 2H)3.31 (s, 2H) 5.14 (s, 2H) 7.29-7.43 (m, 5 H).

N-(2-(Tert-butoxycarbonyl)ethyl)-Gly-OBn (0,030 g, 0.1 mmol) andsuccinimidyl tert-butyloctadecanedioate (described in example 29, 0,050mg, 0.1 mmol) was suspended in dry DMF (1 ml). HOAt (0,014 g, 0.1 mmol)and DIEA (0,21 ml, 1.2 mmol) was added. The yellow reaction mixture wasstirred under nitrogen for 42 h. The reaction mixture was concentrated.The residue was redissolved in AcOEt and washed with 0.1 N HCl (2×),water (1×), dried (Na₂SO₄) and concentrated to give tert-butyloctadecandioyl-N-(2-(tert-butoxycarbonyl)ethyl)-Gly-OBn in 85% yield (55mg).

¹H-NMR (CDCl₃) δ: ppm 1.3 (m, 26H) 1.38 (s, 9H), 1.46 (s, 9 H), 1.6 (m,4H), 2.2 (m, 2H), 2.35 (m, 2 H), 2.65 (m, 2H), 2.85 (s, 2 H) 3.65 (m,2H), 5.15 (s, 2 H) 7.35 (m, 5 H).

Tert-butyl octadecandioyl-N-(2-(tert-butoxycarbonyl)ethyl)-Gly-OBn(0,054 g, 0.08 mmol) was dissolved in THF (2 ml). 10% Palladium oncharcoal was added and the mixture was hydrogenated at 1 atm and RT overthe week-end. The dry reaction mixture was dissolved in AcOEt andfiltered 3 times to remove the carbon. The filtrate was concentrated togive in tert-butyloctadecandioyl-N-(2-(tert-butoxycarbonyl)ethyl)-Gly-OH 80% yield (37mg).

¹H-NMR (CDCl₃) δ: ppm 1.3 (m, 26H) 1.40 (s, 9H), 1.46 (s, 9 H), 1.6 (m,4H), 1.75 (p, 2H), 2.2 (m, 2H), 2.35 (m, 2 H), 2.63 (m, 2H), 2.83 (s, 2H).

Tert-butyl octadecandioyl-N-(2-(tert-butoxyocarbonyl)ethyl)-Gly-OH (0.07mmol) was dissolved in dry THF (2 ml). TSTU (24 mg, 0.08 mmol) and DIPEA(15 uL, 0.08 mmol) was added. The mixture was stirred at RT undernitrogen. After 19 h the reaction had not finished according to TLC(DCM/MeOH 10:1). More DIEA was added (20 uL, 0.11 mmol) and stirring wascontinued. After 42 h the reaction mixture was filtered. The filtratewas diluted with AcOEt and washed with 0.1 N HCl (2×) and brine (1×),dried (Na₂SO₄) and concentrated to a give tert-butyloctadecandioyl-N-(2-(tert-butyxocarbonyl)ethyl)-Gly-OSu as a white solidin 84% yield (36 mg).

¹H-NMR (CDCl₃) δ: ppm 1.3 (m, 26H) 1.40 (m, 2H), 1.44 (s, 9H), 1.46 (s,9 H), 1.58 (m, 2H), 1.73 (p, 2H), 2.2 (t, 2H), 2.60 (t, 2H), 2.8 (m, 6H).

Example31

Synthesis of N^(εB29)—[N—(HOOC(CH₂)₁₄CO)—N-(carboxyethyl)-Gly] des(B30)Human Insulin

This compound was prepared in analogy with example 30, via reaction ofN-(2-(tert-butoxycarbonyl)ethyl)-Gly-OBn with succinimidylhexadecandioate (described in example 4) followed by debenzylation,activation with TSTU, coupling with A1N,B1N-diBOC-Des(B30) human insulinand deprotection by TFA.

MALDI-MS: 6093.0, calculated 6104.1.

Example 32

Synthesis of N^(εB29)—[N—(HOOC(CH₂)₁₄CO)—N-(carboxymethyl)-β-Ala]des(B30) Human Insulin

This compound was prepared in analogy with example 29 via reaction ofN-(tert-butoxycarbonylmethyl)-β-Ala-OBn with succinimidylhexadecandioate (described in example 4) followed by debenzylation,activation with TSTU, coupling with A1N,B1N-diBOC-Des(B30) human insulinand deprotection by TFA.

MALDI-MS: 6097.6, calculated 6104.1.

Example 33

Synthesis of N^(εB29)—[N^(α)-(HOOC(CH₂)₁₁)NHCO(CH₂)₃CO)-γ-L-Glu]des(B30) Human Insulin

This compound was prepared similarly as described in example 1 using(MeOOC(CH₂)₁₁)NHCO(CH₂)₃CO)-Glu(OSu)-OMe, prepared as described below,as acylating agent.

LCMS (electrospray): M+3: 2049, calculated 2050; M+4: 1538, calculated1537.8; M+5: 1231, calculated 1230.4.

MALDI-TOF MS: Calculated: 6147; found: 6153.

Preparation of (MeOOC(CH₂)₁₁)NHCO(CH₂)₃CO)-Glu(OSu)-OMe

MeOH (40 ml) was cooled to 0-5° C., and SOCl₂ (4 ml) was added drop wisewith stirring during 30 minutes. 12-Aminododecanoic acid (3 g, 13.9mmol) was added and the resulting suspension was stirred at 0-5° C.while the ice in cooling bath melted and allowed to warm to roomtemperature during 16 hours. The mixture was filtered and the solid wasdried by suction to afford 2.23 g (60%) of 12-aminododecanoic acidmethyl ester hydrochloride. From the mother liquor a further batch of0.92 g (25%) was isolated.

¹H-NMR (DMSO-d₆) δ: 7.97 (bs, 3H), 3.58 (s, 3H), 2.73 (m, 2H, 2.28 (t,2H), 1.52 (m, 4H), 1.25 (“s”, 14H).

The 12-aminododecanoic acid methyl ester hydrochloride (1 g, 3.8 mmol)was suspended in THF (15 ml) and added glutaric acid anhydride (1.29 g,3.8 mmol) and TEA (0.52 ml, 3.8 mmol) and the resulting mixture(suspension) was stirred at room temperature for 16 hours. Water (75 ml)was added gradually. After 25 ml, a solution was obtained and later asuspension appeared. The mixture was stirred at room temperature for 1hour and filtered. The solid was washed with water and dried in vacuo.This afforded 1.02 g (80%) of 12-(4-carboxybutyrylamino)dodecanoic acidmethyl ester.

¹H-NMR (DMSO-d₆) δ: 12 (bs, 1H), 7.73 (t, 1H), 3.57 (s, 3H), 3.00 (q,2H), 2.28 (t, 2H), 2.18 (t, 2H), 2.06 (t, 2H), 1.69 (p, 2H), 1.50 (p,2H), 1.36 (p, 2H),1.23 (“s”, 14H).

The 12-(4-carboxybutyrylamino)dodecanoic acid methyl ester (0.33 g, 0.95mmol) was dissolved in a mixture of THF and DMF (2:1, 6 ml), and addedDIEA (0.178 ml, 1.04 mmol). The mixture was cooled to 0-5° C. and TSTU(0.314 g, 1.04 mmol) was added. The mixture was stirred at 0-5° C. for 1hour and at room temperature for 16 hours. The mixture was concentratedto dryness in vacuo. The residue (OSu-activated12-(4-carboxybutyrylamino)dodecanoic acid methyl ester) was dissolved inDMF (10 ml) and added DIEA (0.24 ml, 1.4 mmol) and H-Glu-OMe (0.168 g,1.04 mmol) and the mixture was stirred at room temperature for 16 hours.The mixture was concentrated in vacuo and the residue was dissolved inAcOEt (100 ml) and washed with 0.2M hydrochloric acid (3×50 ml). Theorganic phase was dried (Na₂SO₄) and concentrated in vacuo. Thisafforded 0.358 g (78%) of (MeOOC(CH₂)₁₁)NHCO(CH₂)₃CO)-Glu-OMe.

¹H-NMR (DMSO-d₆), δ: 12 (bs, 1H), 8.22 (d, 1H), 7.73 (t, 1H), 4.24 (m,1H), 3.61 (s, 3H), 3.57 (s, 3H), 3.00 (q, 2H), 2.27 (m, 4H), 2.10 (t,2H), 2.04 (t, 2H), 1.9 (m, 1H), 1.8 (m, 1H), 1.68 (t, 2H), 1.50 (m, 2H),1.36 (m, 2H), 1.23 (“s”, 14H).

(MeOOC(CH₂)₁₁)NHCO(CH₂)₃CO)-Glu-OMe (0.36 g, 0.36 mmol) was dissolved inTHF (10 ml) and cooled to 0-5° C. DIEA (0.13 ml) and TSTU (0.129 g, 0.43mmol) were added and the mixture was stirred at 0-5° C. for some hoursand at room temperature for 3 days. The mixture was concentrated invacuo. The residue was dissolved in AcOEt (100 ml) and washed with 0.2Nhydrochloric acid (3×50 ml) and saturated aqueous NaHCO₃ (3×100 ml).Drying (Na₂SO₄) and concentration in vacuo afforded 0.17 g (84%) of(MeOOC(CH₂)₁₁)NHCO(CH₂)₃CO)-Glu(OSu)-OMe.

¹H-NMR (DMSO-d₆), selected peaks, δ: 8.27 (d, 1H), 7.72 (t, 1H), 4.31(m, 1H), 3.63 (s, 3H), 3.57 (s, 3H), 3.00 (q, 2H), 2.81 (s, 4H), 2.28(t, 2H), 2.12 (t, 2H), 2.05 (t, 2H), 1.70 (m, 2H), 1.50 (m, 2H), 1.35(m, 2H), 1.23 (“s”, 14H).

Example 34

Synthesis of N^(εB29)—[N^(α)-(HOOC(CH₂)₁₁)NHCO(CH₂)₂CO)-γ-L-Glu]des(B30) Human Insulin

This compound was prepared similarly as described in example 1 using(MeOOC(CH₂)₁₁)NHCO(CH₂)₂CO)-Glu(OSu)-OMe, which in turn was preparedsimilarly as described in example 33 using succinic acid anhydrideinstead of glutaric acid anhydride, as acylating agent.

MALDI-TOF MS: Calculated: 6133; found: 6134.

Example 35

Synthesis of N^(εB29)—[N^(α)-(HOOC(CH₂)₁₆CO)]-Gly-γ-L-Glu des(B30) HumanInsulin

This compound was prepared similarly as described in example 4 usingtert-butyl octadecandioyl-Gly-Glu(OSu)-O^(t)Bu, prepared as describedbelow as acylating agent.

MALDI-TOF MS: Calculated: 6189; found: 6191.

Preparation of Tert-Butyl Octadecandioyl-Gly-Glu(OSu)-OtBu

Z-Gly-OH (1.0 g, 4.78 mmol) was added THF (10 ml), DIEA (0.98 ml, 5.74mmol), and TSTU (1.7 g, 5.74 mmol) and the resulting mixture was stirredat room temperature for 2 hours. AcOEt (100 ml) was added and themixture was washed with 0.2N hydrochloric acid (100 ml) and water (2×100ml). The organic phase was dried (Na₂SO₄) and concentrated in vacuo toafford 1.34 g (92%) of Z-Gly-OSu as an oil.

¹H-NMR (CDCl₃), δ: 7.35 (s, 5H), 5.32 (t, 1H), 5.15 (s, 2H), 4.35 (d,2H), 2.83 (s, 4H).

Z-Gly-OSu (1.3 g, 4.25 mmol) was dissolved in DMF (15 ml) and DIEA (1.82ml, 10.6 mmol) and H-Glu-O^(t)Bu (0.949 g, 4.67 mmol) were added and theresulting mixture was stirred at room temperature for 16 hours. AcOEt(100 ml) was added and the mixture was washed with 0.2N hydrochloricacid (100 ml) and water (2×100 ml). The organic phase was dried (Na₂SO₄)and concentrated in vacuo to afford 1.7 g (quant.) of Z-Gly-Glu-O^(t)Buas an oil.

¹H-NMR (CDCl₃), δ: 7.33 (s, 5H), 7.1 (d, 1H), 5.80 (t, 1H), 5.12 (s,2H), 4.53 (m, 1H), 3.90 (d, 2H), 2.36 (t, 2H), 2.22 (m,1H), 1.95 (m,1H), 1.45 (s, 9H).

Z-Gly-Glu-O^(t)Bu (1.7 g, 4.3 mmol) was dissolved in 1,4-dioxane (15 ml)and under N₂ 10% palladium black (0.6 g) was added. The mixture washydrogenated at atmospheric pressure for 5 hours. The mixture wasfiltered and the palladium was stirred with water (200 ml) for 2 hours,filtered and the filtrate was lyophilized. This afforded 0.65 g (58%) ofH-Gly-Glu-O^(t)Bu.

¹H-NMR (DMSO-d₆), selected peaks, δ: 8.31 (d, 1H), 2.20 (t, 2H), 1.91(m, 1H), 1.80 (m, 1H), 1.40 (s, 9H).

H-Gly-Glu-O^(t)Bu (0.15 g, 0.58 mmol) was suspended in DMF (5 ml) andDIEA (0.15 ml, 0.86 mmol) and succinimidyl tert-butyl octadecandioate(0.27 g, 0.58 mmol) were added and the resulting mixture was stirred atroom temperature for 16 hours. AcOEt (50 ml) was added and the mixturewas washed with 0.2N hydrochloric acid (100 ml) and water (3×100 ml).The organic phase was dried (Na₂SO₄) and concentrated in vacuo to afford0.34 g (quant.) of tert-butyl octadecandioyl-Gly-Glu-O^(t)Bu.

¹H-NMR (CDCl₃), δ: 7.11 (d, 1H), 6.55 (t, 1H), 4.55 (dt, 1H), 4.00 (dq,2H), 2.40 (t, 2H), 2.26 (t, 2H), 2.20 (t, 4H), 2.00 (m, 1H), 1.57-1.65(m, 5H), 1.47 (s, 9H), 1.44 (s, 9H), 1.25 (“s”, 22H, overlap with HDO).

Tert-butyl octadecandioyl-Gly-Glu-O^(t)Bu (0.32 g, 0.52 mmol) wasdissolved in THF (5 ml) and added DIEA (0.11 ml, 0.63 mmol) and TSTU(0.19 g, 0.63 mmol) and the resulting mixture was stirred at roomtemperature under N₂ for 3 days. AcOEt (100 ml) was added and themixture was washed with 0.15N hydrochloric acid (100 ml) and water(3×100 ml). The organic phase was dried (Na₂SO₄) and concentrated invacuo to afford 0.3 g (81%) of tert-butyloctadecandioyl-Gly-Glu(OSu)-O^(t)Bu.

¹H-NMR (CDCl₃), selected peaks, δ: 6.89 (d,1H), 6.44 (t, 1H), 4.60(m,1H), 3.95 (dq, 2H), 2.86 (s, 4H), 2.68 (q, 2H), 2.24 (t, 2H), 2.20(t, 4H), 1.57-1.65 (m, 5H), 1.48 (s, 9H), 1.44 (s, 9H), 1.25 (“s”, 22H,overlap with HDO).

Example 36

Synthesis of N^(εB29)—[N—(HOOC(CH₂)₁₄CO)—N-(2-carboxyethyl)-β-Ala] desB30 Human Insulin

This compound was prepared in analogy with example 1, via coupling of15-[[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonyl)-ethyl]-(2-methoxycarbonyl-ethyl)-carbamoyl]-pentadecanoicacid methyl ester with Des(B30) human insulin and deprotection by NaOH.

LC-MS: M+4. 1530.3, calculated 1529.5

Preparation of Methyl HexadecandioylN-(2-(methoxycarbonyl)ethyl)-β-Ala-OSu

H-β-Ala-OMe hydrochloride (5.45 g, 39 mmol) was dissolved in DMSO (100ml) and tert-butyl acrylate (5.71 ml, 39 mmol) and DIEA (13.4 ml, 78mmol) were added, and the resulting mixture was stirred at roomtemperature for 6 days. The mixture was partitioned between water (500ml) and AcOEt (2×250 ml). The combined organic phases were washed withsaturated aqueous NH₄Cl, dried (MgSO₄) and concentrated in vacuo. Thisafforded 7.24 g (80%) of N-(2-(methoxycarbonyl)ethyl)-β-Ala-OtBu.

¹H-NMR (CDCl₃) δ: 3.58 (s, 3H), 2.72 (t, 2H), 2.67 (t, 2H), 2.41 (t,2H), 2.29 (t, 2H),1.39 (s, 9H).

Hexadecanedioic acid monomethyl ester (150 mg, 0.5 mmol) was dissolvedin DMF (5 mL). HOAt (102 mg, 0.75 mmol) and EDAC (143 mg, 0.75 mmol) wasadded and the reaction was stirred at 50° C. for 1 hour. After coolingto room temperature, DIEA (0.256 mL, 1.5 mmol) andN-(2-(methoxycarbonyl)ethyl)-β-Ala-OtBu (139 mg, 0.6 mmol) was added.The reaction was stirred overnight at room temperature. The mixture waspartitioned between water (2×50 mL) and AcOEt (100 mL). The organicphase was dried (Na₂SO₄) and concentrated in vacuo. to an oil. DCM (10mL) and TFA (10 mL) was added and the mixture was stirred for 1 hour atroom temperature, solvent removed in vacuo to afford 170 mg (87%) ofmethyl hexadecandioyl N-(2-(methoxycarbonyl)ethyl-β-Ala-OH.

LC-MS: 458 (M+1).

Methyl hexadecandioyl N-(2-(methoxycarbonyl)ethyl-β-Ala-OH (161 mg,0.351 mmol) was dissolved in THF (10 mL) DIEA (0.073 mL, 0.42 mmol) andTSTU (127 mg, 0.42 mmol) was added. The mixture was stirred while cooledon an icebath for 30 min, followed by stirring for 2 hours at roomtemperature. The mixture was partitioned between and AcOEt (100 mL) andaquoues HCl (0.2 N, 2×80 mL). The organic phase was dried (Na₂SO₄) andconcentrated in vacuo. This afforded 140 mg (72%) of methylhexadecandioyl N-(2-(methoxycarbonyl)ethyl)-β-Ala-OSu as an oil.

LC-MS: 555 (M+1).

Example 37

Synthesis of N^(εB29)—[N—(HOOC(CH₂)₁₆CO)—N-(2-carboxyethyl)-β-Ala] desB30 Human Insulin

This compound was prepared in analogy with example 1 and 36 via couplingof methyl octadecandioyl N-(2-(methoxycarbonyl)ethyl)-β-Ala-OSu withDes(B30) human insulin and deprotection by NaOH.

Preparation of Methyl OctadecandioylN-(2-(Methoxycarbonyl)Ethyl)-β-Ala-OSu

This compound was synthesized in analogy with methyl hexadecandioylN-(2-(methoxycarbonyl)ethyl)-β-Ala-OSu using octadecanedioic acid monomethyl ester.

MALDI-TOF MS: Calculated 6146; found: 6151

LC-MS: 583 (M+1)

PHARMACOLOGICAL METHODS

Assay (I)

Insulin Receptor Binding Of The Insulin Derivatives Of The Invention

The affinity of the insulin analogues of the invention for the humaninsulin receptor was determined by a SPA assay (Scintillation ProximityAssay) microtiterplate antibody capture assay. SPA-PVT antibody-bindingbeads, anti-mouse reagent (Amersham Biosciences, Cat No. PRNQ0017) weremixed with 25 ml of binding buffer (100 mM HEPES pH 7.8; 100 mM sodiumchloride, 10 mM MgSO₄, 0.025% Tween-20). Reagent mix for a singlePackard Optiplate (Packard No. 6005190) is composed of 2.4 μl of a1:5000 diluted purified recombinant human insulin receptor—exon 11, anamount of a stock solution of A14 Tyr[¹²⁵I]—human insulin correspondingto 5000 cpm per 100 μl of reagent mix, 12 μl of a 1:1000 dilution of F12antibody, 3 ml of SPA-beads and binding buffer to a total of 12 ml. Atotal of 100 μl was then added and a dilution series is made fromappropriate samples. To the dilution series was then added 100 μl ofreagent mix and the samples were incubated for 16 hours while gentlyshaken. The phases were the then separated by centrifugation for 1 minand the plates counted in a Topcounter. The binding data were fittedusing the nonlinear regression algorithm in the GraphPad Prism 2.01(GraphPad Software, San Diego, Calif.).

Preparation of Monoclonal mIR Antibodies

Specific antibodies (F12) were produced by monoclonal technique: RBFmice were immunized by injecting 50 μg of purified mIR in FCAsubcutaneously followed by two injections with 20 μg of mIR in FIA.Highresponder mice were boosted intravenously with 25 μg of mIR and thespleens were harvested after 3 days. Spleen cells were fused with themyeloma Fox cell line (Kohler, G & Milstein C. (1976), European J.Immunology, 6:511-19; Taggart RT et al (1983), Science 219:1228-30).Supernatants were screened for antibody production in a mIR specificELISA. Positive wells were cloned and tested in Western blotting.

TABLE 2 Receptor binding Product (% of human insulin) Human insulin 100N^(εB29)—(N—HOOC(CH₂)₁₄CO-γ-Glu) des(B30) human insulin 26N^(εB29)—(N—HOOC(CH₂)₁₆CO-γ-Glu) des(B30) human insulin 9.2N^(εB29)—(N—HOOC(CH₂)₁₆CO-γ-Glu-N-(γ-Glu) des(B30) human insulin 11N^(εB29)—(N-(Asp-OC(CH₂)₁₆CO)-γ-Glu) des(B30) human insulin 13N^(εB29)—(N-(Glu-OC(CH₂)₁₄CO-γ-Glu) des(B30) human insulin 13N^(εB29)—(N-(Glu-OC(CH₂)₁₄CO—) des(B30) human insulin 9.4N^(εB29)—(N—HOOC(CH₂)₁₆CO-α-Glu)-N-(β-Asp) des(B30) human insulin 11N^(εB29)—(N-(Gly-OC(CH₂)₁₃CO-γ-Glu) des(B30) human insulin 22N^(εB29)—(N-(Sar-OC(CH₂)₁₃CO-γ-Glu) des(B30) human insulin 20N^(εB29)—(N—HOOC(CH₂)₁₆CO-α-L-Asp)-N-(β-L-Asp) des(B30) human insulin 14N^(εB29)—(N-(Gly-OC(CH₂)₁₄CO-γ-Glu) des(B30) human insulin 32N^(εB29)—(N—HOOC(CH₂)₁₅CO-γ-L-Glu) des(B30) human insulin 4N^(εB29)—(N—HOOC(CH₂)₁₄CO-β-L-Asp) des(B30) human insulin 16 0525N^(εB29)—(N—HOOC(CH₂)₁₄CO-β-D-Asp) des(B30) human insulin 37N^(εB29)—(N—HOOC(CH₂)₁₃CO-β-L-Glu) des(B30) human insulin 15N^(εB29)—(N—HOOC(CH₂)₁₃CO-β-L-Asp) des(B30) human insulin 11N^(εB29)—(N—HOOC(CH₂)₁₆CO-δ-L-Aad) des(B30) human insulin 7N^(εB29)—(N—HOOC(CH₂)₁₆CO-γ-D-Glu) des(B30) human insulin 13N^(εB29)—(N—HOOC(CH₂)₁₅CO-β-L-Asp) des(B30) human insulin 5.4N^(εB29)—(N—HOOC(CH₂)₁₆CO-α-L-Asp) des(B30) human insulin 13N^(εB29)—(N—HOOC(CH₂)₁₆CO-α-L-Glu) des(B30) human insulin 16N^(εB29)—(N—(HOOC(CH₂)₁₄CO-ε-L-LysCO—) des(B30) human insulin 5.7N^(εB29)—(N—HOOC(CH₂)₁₆CO-β-L-Asp) des(B30) human insulin 11N^(εB29)—(N-(Gly-OC(CH₂)₁₆CO-γ-L-Glu) des(B30) human insulin 9.1N^(εB29)—[N—(HOOC(CH₂)₁₆CO)—N-(carboxymethyl)-β-Ala] des(B30) humaninsulin 9.4 N^(εB29)—[N^(α)—(HOOC(CH₂)₁₁)NHCO(CH₂)₃CO)-γ-L-Glu] des(B30)human insulin 46Assay (II)Potency of the Insulin Derivatives of the Invention Relative to HumanInsulin

Sprague Dawley male rats weighing 238-383 g on the experimental day wereused for the clamp experiment. The rats had free access to feed undercontrolled ambient conditions and were fasted overnight (from 3 pm)prior to the clamp experiment.

Experimental Protocol

The rats were acclimatized in the animal facilities for at least 1 weekprior to the surgical procedure. Approximately 1 week prior to the clampexperiment Tygon catheters were inserted under halothane anaesthesiainto the jugular vein (for infusion) and the carotid artery (for bloodsampling) and exteriorised and fixed on the back of the neck. The ratswere given Streptocilin vet. (Boehringer Ingelheim; 0.15 ml/rat, i.m.)post-surgically and placed in an animal care unit (25° C.) during therecovery period. In order to obtain analgesia, Anorphin (0.06 mg/rat,s.c.) was administered during anaesthesia and Rimadyl (1.5 mg/kg, s.c.)was administered after full recovery from the anaesthesia (2-3 h) andagain once daily for 2 days.

The clamp technique employed was adapted from (1). At 7 am on theexperimental day overnight fasted (from 3 pm the previous day) rats wereweighed and connected to the sampling syringes and infusion system(Harvard 22 Basic pumps, Harvard, and Perfectum Hypodermic glasssyringe, Aldrich) and then placed into individual clamp cages where theyrested for ca. 45 min before start of experiment. The rats were able tomove freely on their usual bedding during the entire experiment and hadfree access to drinking water. After a 30 min basal period during whichplasma glucose levels were measured at 10 min intervals, the insulinderivative to be tested and human insulin (one dose level per rat, n=6-7per dose level) were infused (i.v.) at a constant rate for 300 min.Plasma glucose levels were measured at 10 min intervals throughout andinfusion of 20% aqueous glucose was adjusted accordingly in order tomaintain euglyceamia. Samples of re-suspended erythrocytes were pooledfrom each rat and returned in about ½ ml volumes via the carotidcatheter.

On each experimental day, samples of the solutions of the individualinsulin derivatives to be tested and the human insulin solution weretaken before and at the end of the clamp experiments and theconcentrations of the peptides were confirmed by HPLC. Plasmaconcentrations of rat insulin and C-peptide as well as of the insulinderivative to be tested and human insulin were measured at relevant timepoints before and at the end of the studies. Rats were killed at the endof experiment using a pentobarbital overdose.

Test compounds and doses: Insulins to be tested were diluted from astock solution containing 97 μM of the insulin derivative in 5 mMphosphate pH 7.7. The final concentration in the solution ready for usewas 0.45 μM of the insulin derivative, 5 mM of phosphate, 100 mM ofsodium chloride, 0.007% of polysorbate 20. The pH was 7.7 and the i.v.infusion rate was 15 and 20 pmol-min⁻¹−kg⁻¹.

A stock solution of human insulin that was used as reference compoundwas formulated in a similar medium and infused i.v. at 6, 15 or 30pmol·min⁻¹−kg⁻¹.

Both stock solutions were stored at −20 C. and thawed overnight at 4° C.before use. The solutions were gently turned upside down several times15 min before they were transferred to the infusion syringes.

TABLE 3 Potency relative Insulin derivative to human insulinN^(εB29)—(N—HOOC(CH₂)₁₄CO-γ-Glu) des(B30) human insulin >50%N^(εB29)—(N—HOOC(CH₂)₁₆CO-γ-Glu) des(B30) human insulin >50%N^(εB29)—(N—HOOC(CH₂)₁₆CO-γ-Glu-N-(γ-Glu) des(B30) human insulin >50%N^(εB29)—(N—HOOC(CH₂)₁₄CO-β-L-Asp) des(B30) human insulin >50%N^(εB29)—(N-(Gly-OC(CH₂)₁₄CO-γ-Glu) des(B30) human insulin >50%N^(εB29)—(N—HOOC(CH₂)₁₄CO-β-L-Asp) des(B30) human insulin >50%Assay (III)Determination in Pigs of T_(50%) of the Insulin Derivatives of theInvention

T_(50%) is the time when 50% of an injected amount of the A14 Tyr[¹²⁵I]labelled derivative of an insulin to be tested has disappeared from theinjection site as measured with an external γ-counter.

The principles of laboratory animal care were followed, Specificpathogen-free LYYD, non-diabetic female pigs, cross-breed of DanishLandrace, Yorkshire and Duroc, were used (Holmenlund, Haarloev, Denmark)for pharmacokinetic and pharmacodynamic studies. The pigs wereconscious, 4-5 months of age and weighing 70-95 kg. The animals werefasted overnight for 18 h before the experiment.

Formulated preparations of insulin derivatives labelled in Tyr^(A14)with ¹²⁵I were injected sc. in pigs as previously described (Ribel, U.,Jørgensen, K, Brange, J, and Henriksen, U. The pig as a model forsubcutaneous insulin absorption in man. Serrano-Rios, M and Lefèbvre, P.J. 891-896.1985. Amsterdam; New York; Oxford, Elsevier SciencePublishers. 1985 (Conference Proceeding)).

At the beginning of the experiments a dose of 60 nmol of the insulinderivative according to the invention (test compound) and a dose of 60nmol of insulin detemir (both ¹²⁵I labelled in Tyr A14) were injected attwo separate sites in the neck of each pig.

The disappearance of the radioactive label from the site of sc.injection was monitored using a modification of the traditional externalgamma-counting method (Ribel, U. Subcutaneous absorption of insulinanalogues. Berger, M. and Gries, F. A. 70-77 (1993). Stuttgart; NewYork, Georg Thime Verlag (Conference Proceeding)). With this modifiedmethod it was possible to measure continuously the disappearance ofradioactivity from a subcutaneous depot for several days using cordlessportable device (Scancys Laboratorieteknik, Vaerløse, DK-3500, Denmark).The measurements were performed at 1-min intervals, and the countedvalues were corrected for background activity.

In Table 4, the column “test/detemir” shows the T_(50%) found for eachof the compounds tested (“test”) and the T_(50%) found for insulindetemir (“detemir”) in the same experiment.

TABLE 4 T_(50%), hours Insulin derivative test/detemirN^(εB29)—(N—HOOC(CH₂)₁₄CO-γ-Glu) des(B30) human insulin 9.0/9.5N^(εB29)—(N—HOOC(CH₂)₁₆CO-γ-Glu) des(B30) human insulin 10.6/9.7 N^(εB29)—(N—HOOC(CH₂)₁₆CO-γ-Glu-N-(γ-Glu) des(B30) human insulin 7.8/7.4N^(εB29)—(N-(Asp-OC(CH₂)₁₆CO)-γ-Glu) des(B30) human insulin 3.5/7.4N^(εB29)—(N-(Asp-OC(CH₂)₁₆CO—) des(B30) human insulin 4.1/7.4N^(εB29)—(N—HOOC(CH₂)₁₆CO-α-Glu)-N-(β-Asp) des(B30) human insulin8.7/9.1

The invention claimed is:
 1. An insulin derivative which is a naturallyoccurring insulin or an analogue thereof which has a side chain attachedto the α-amino group of the N-terminal amino acid residue of the B chainor to the ε-amino group of a Lys residue present in the B chain of theparent insulin, the side chain being of the general formula:—W—X—Y—Z wherein W is two amino acid residues linked together via amidebonds, which chain—via an amide bond—is linked to the α-amino group ofthe N-terminal amino acid residue of the B chain or to the ε-amino groupof a Lys residue present in the B chain of the parent insulin, the aminoacid residues of W being selected from the group of amino acid residueshaving a neutral side chain and amino acid residues having a carboxylicacid group in the side chain so that W has at least one amino acidresidue which has a carboxylic acid group in the side chain; X is —CO—;Y is —(CH₂)_(m)— where m is an integer in the range of 6 to 32; and Z is—COOH; and any Zn²⁺complexes thereof.
 2. An insulin derivative accordingto claim 1, wherein side chain —W—X—Y—Z is attached to the ε-amino groupof a Lys residue present in the B chain of the parent insulin.
 3. Aninsulin derivative according to claim 1, wherein W is a chain composedof two α-amino acid residues of which one has from 4 to 10 carbon atomsand a free carboxylic acid group while the other has from 2 to 11 carbonatoms but no free carboxylic acid group.
 4. An insulin derivativeaccording to claim 3 wherein W is selected from the group consisting ofα-Asp-Gly; Gly-α-Asp; β-Asp-Gly; Gly-β-Asp; α-Glu-Gly; Gly-α-Glu;γ-Glu-Gly; Gly-γ-Glu; α-hGlu-Gly; Gly-α-hGlu; δ-hGlu-Gly; andGly-δ-hGlu.
 5. An insulin derivative according to claim 1, wherein W isa chain composed of two α-amino acid residues, independently having from4 to 10 carbon atoms, and both having a free carboxylic acid group. 6.An insulin derivative according to claim 5, wherein W is selected fromthe group consisting of α-Asp-α-Asp; α-Asp-α-Glu; α-Asp-α-hGlu;α-Asp-β-Asp; α-Aspγ-Glu; α-Asp-δ-hGlu;β-Asp-α-Asp; β-Asp-α-Glu;β-Asp-α-hGlu; β-Asp-β-Asp; β-Asp-γ-Glu; β-Asp-δ-hGlu; α-Glu-α-Asp;α-Glu-α-Glu; α-Glu-α-hGlu; α-Glu-β-Asp; α-Glu-γ-Glu; α-Glu-δ-hGlu;γ-Glu-α-Asp; γ-Glu-α-Glu; γ-Glu-α-hGlu; γ-Glu-β-Asp; γ-Glu-γ-Glu;γ-Glu-δ-hGlu; α-hGlu-α-Asp; α-hGlu-α-Glu; α-hGlu-α-hGlu; α-hGlu-β-Asp;α-hGlu-γ-Glu; α-hGlu-δ-hGlu; δ-hGlu-α-Asp; δ-hGlu-α-Glu; δ-hGlu-α-hGlu;δ-hGlu-β-Asp; δ-hGlu-γ-Glu; and δ-hGlu-δ-hGlu.
 7. An insulin derivativeaccording to claim 1, wherein Y is —(CH₂)_(m)—where m is an integer inthe range of from 12-16.
 8. An insulin derivative according to claim 1,wherein the parent insulin is des(B30) human insulin.
 9. An insulinderivative according to claim 1, wherein the insulin derivative isselected from the group consisting ofN^(εB29)—(N-HOOC(CH₂)₁₆CO-γ-Glu-N-(γ-Glu) des(B30) human insulin;N^(εB29)—(N-HOOC(CH₂)₁₆CO-α-Glu)-N-(β-Asp) des(B30) human insulin; andN^(εB29)—(N-HOOC(CH₂)₁₆CO-α-Glu)-N-(AspAsp) des(B30) human insulin. 10.A zinc complex of an insulin derivative according to claim 1, whereineach insulin hexamer in said complex binds two zinc ions.
 11. A zinccomplex of an insulin derivative according to claim 1, wherein eachinsulin hexamer in said complex binds three zinc ions.
 12. A zinccomplex of an insulin derivative according to claim 1, wherein eachinsulin hexamer in said complex binds four zinc ions.
 13. Apharmaceutical composition for the treatment of diabetes in a patient inneed of such treatment, said composition comprising a therapeuticallyeffective amount of an insulin derivative according to claim 1 togetherwith a pharmaceutically acceptable carrier.
 14. A pharmaceuticalcomposition for the treatment of diabetes in a patient in need of suchtreatment, said composition comprising a therapeutically effectiveamount of an insulin derivative according to claim 1 in mixture with aninsulin or an insulin analogue which has a rapid onset of action,together with a pharmaceutically acceptable carrier.
 15. A method oftreating diabetes, said method comprising administering to a patient inneed of such a treatment a therapeutically effective amount of aninsulin derivative according to claim 1 together with a pharmaceuticallyacceptable carrier.
 16. A method of treating diabetes, said methodcomprising administering to a patient in need of such a treatment atherapeutically effective amount of an insulin derivative according toclaim 1 in mixture with an insulin or an insulin analogue which has arapid onset of action, together with a pharmaceutically acceptablecarrier.